Chemically modified single-stranded RNA-editing oligonucleotides

ABSTRACT

The invention relates to antisense oligonucleotides that are capable of bringing about specific editing of a target nucleotide (adenosine) in a target RNA sequence in a eukaryotic cell, wherein said oligonucleotide does not, in itself, form an intramolecular hairpin or stem-loop structure, and wherein said oligonucleotide comprises a non-complementary nucleotide in a position opposite to the nucleotide to be edited in the target RNA sequence.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/329,787, filed Mar. 1, 2019, which is a § 371 National StageApplication of PCT/EP2017/071912, filed Aug. 31, 2017, which claimspriority to and the benefit of United Kingdom patent application No.1614858.7, filed Sep. 1, 2016, United Kingdom patent application No.1616374.3, filed Sep. 27, 2016, United Kingdom patent application No.1621467.8, filed Dec. 16, 2016, United Kingdom patent application No.1703034.7, filed Feb. 24, 2017, and United Kingdom patent applicationNo. 1707508.6, filed May 10, 2017, the entire disclosures of each ofwhich are incorporated herein by reference for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name 4430_0030006_Sequence_Listing_ST25.K Size: 9,949 bytes;and Date of Creation: Mar. 7, 2023) filed with the application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of medicine. In particular, itrelates to the field of RNA editing, whereby an RNA sequence is targetedby a single-stranded antisense oligonucleotide to correct a geneticmutation and/or to alter the sequence of a specific target RNA. More inparticular, the invention relates to chemical modifications of thesingle-stranded RNA-editing oligonucleotides that render theoligonucleotides more stable and increase their RNA-editing efficiency.

BACKGROUND OF THE INVENTION

RNA editing is a natural process through which eukaryotic cells alterthe sequence of their RNA molecules, often in a site-specific andprecise way, thereby increasing the repertoire of genome encoded RNAs byseveral orders of magnitude. RNA editing enzymes have been described foreukaryotic species throughout the animal and plant kingdoms, and theseprocesses play an important role in managing cellular homeostasis inmetazoans from the simplest life forms such as Caenorhabditis elegans,to humans. Examples of RNA editing are adenosine (A) to inosine (I) andcytidine (C) to uridine (U) conversions through enzymes called adenosinedeaminase and cytidine deaminase, respectively. The most extensivelystudied RNA editing system is the adenosine deaminase enzyme.

Adenosine deaminase is a multidomain protein, comprising a recognitiondomain and a catalytic domain. The recognition domain recognizes aspecific double-stranded RNA (dsRNA) sequence and/or conformation,whereas the catalytic domain converts an adenosine into an inosine in anearby, more or less predefined, position in the target RNA, bydeamination of the nucleobase. Inosine is read as guanosine by thetranslational machinery of the cell, meaning that, if an editedadenosine is in a coding region of an mRNA or pre-mRNA, it can recodethe protein sequence.

A-to-I conversions may also occur in 5′ non-coding sequences of a targetmRNA, creating new translational start sites upstream of the originalstart site, which gives rise to N-terminally extended proteins. Editingevents can also occur at 3′ UTRs and affect miRNA-based regulation andpolyadenylation. In addition, A-to-I conversions may take place insplice elements in introns or exons in pre-mRNAs, thereby altering thepattern of splicing. As a consequence, exons may be included or skipped.The adenosine deaminases are part of a family of enzymes referred to asAdenosine Deaminases acting on RNA (ADAR), including human deaminaseshADAR1, hADAR2 and hADAR3.

The use of oligonucleotides to edit a target RNA applying adenosinedeaminase is known in the art. Montiel-Gonzalez et al. (PNAS 2013,110(45):18285-18290) described the editing of a target RNA using agenetically engineered fusion protein, comprising an adenosine deaminasedomain of the hADAR2 protein, fused to a bacteriophage lambda N protein,which recognises the boxB RNA hairpin sequence. The natural dsRNAbinding domains of hADAR2 had been removed to eliminate the substraterecognition properties of the natural ADAR and replace it by the boxBrecognition domain of lambda N-protein. The authors created an antisenseoligonucleotide comprising a ‘guide RNA’ part that is complementary tothe target sequence for editing, fused to a boxB portion for sequencespecific recognition by the N-domain-deaminase fusion protein. By doingso, it was elegantly shown that the guide RNA oligonucleotide faithfullydirected the adenosine deaminase fusion protein to the target site,resulting in guide RNA-directed site-specific A-to-I editing of thetarget RNA. The guide RNAs disclosed in Montiel-Gonzalez et al. (2013)are longer than 50 nucleotides in length. A disadvantage of this methodin a therapeutic setting is the need for a fusion protein consisting ofthe boxB recognition domain of bacteriophage lambda N-protein,genetically fused to the adenosine deaminase domain of a truncatednatural ADAR protein. It requires target cells to be either transducedwith the fusion protein, which is a major hurdle, or that target cellsare transfected with a nucleic acid construct encoding the engineeredadenosine deaminase fusion protein for expression. The latterrequirement constitutes no minor obstacle when editing is to be achievedin a multicellular organism, such as in therapy against human disease tocorrect a genetic disorder.

Vogel et al. (2014. Angewandte Chemie Int Ed 53:267-271) disclosedediting of RNA coding for eCFP and Factor V Leiden, using abenzylguanosine substituted guide RNA and a genetically engineeredfusion protein, comprising the adenosine deaminase domains of ADAR1 or 2(lacking the dsRNA binding domains) genetically fused to a SNAP-tagdomain (an engineered 06-alkylguanosi ne-DNA-alkyl transferase).Although the genetically engineered artificial deaminase fusion proteincould be targeted to a desired editing site in the target RNAs in HeLacells in culture, through its SNAP-tag domain which is covalently linkedto a guide RNA through a 5′-terminal 06-benzylguanosine modification,this system suffers from similar drawbacks as the genetically engineeredADARs described by Montiel-Gonzalez et al. (2013), in that it is notclear how to apply the system without having to genetically modify theADAR first and subsequently transfect or transduct the cells harboringthe target RNA, to provide the cells with this genetically engineeredprotein. Clearly, this system is not readily adaptable for use inhumans, e.g. in a therapeutic setting.

Woolf et al. (1995. Proc Natl Acad Sci USA 92:8298-8302) disclosed asimpler approach, using relatively long single-stranded antisense RNAoligonucleotides (25-52 nucleotides in length) wherein the longeroligonucleotides (34-mer and 52 mer) could promote editing of the targetRNA by endogenous ADAR because of the double-stranded nature of thetarget RNA and the hybridizing oligonucleotide. The oligonucleotides ofWoolf et al. (1995) that were 100% complementary to the target RNAsequences only appeared to function in cell extracts or in amphibian(Xenopus) oocytes by microinjection, and suffered from severe lack ofspecificity: nearly all adenosines in the target RNA strand that wascomplementary to the antisense oligonucleotide were edited. Anoligonucleotide, 34 nucleotides in length, wherein each nucleotidecomprised a 2′-O-methyl modification, was tested and shown to beinactive in Woolf et al. (1995). In order to provide stability againstnucleases, a 34-mer RNA, modified with 2′-O-methyl-modifiedphosphorothioate nucleotides at the 5′- and 3′-terminal 5 nucleotides,was also tested. It was shown that the central unmodified region of thisoligonucleotide could promote editing of the target RNA by endogenousADAR, with the terminal modifications providing protection againstexonuclease degradation. Woolf et al. (1995) did not achieve deaminationof a specific target adenosine in the target RNA sequence. Nearly alladenosines opposite an unmodified nucleotide in the antisenseoligonucleotide were edited (therefore nearly all adenosines oppositenucleotides in the central unmodified region, if the 5′- and 3′-terminal5 nucleotides of the antisense oligonucleotide were modified, or nearlyall adenosines in the target RNA strand if no nucleotides weremodified). ADAR acts on any double stranded RNA (dsRNA). Through aprocess sometimes referred to as ‘promiscuous editing’, the enzyme willedit multiple adenosines in a dsRNA. Hence, there is a need for methodsand means that circumvent such promiscuous editing and that only targetspecified adenosines in a target RNA sequence. Vogel et al. (2014)showed that such off-target editing can be suppressed by using2′-O-methyl-modified nucleotides in the oligonucleotide at positionsopposite to the adenosines that should not be edited, and use anon-modified nucleotide directly opposite to the specifically targetedadenosine on the target RNA. However, the specific editing effect at thetarget nucleotide has not been shown to take place in that articlewithout the use of recombinant ADAR enzymes that specifically formcovalent bonds with the antisense oligonucleotide.

WO 2016/097212 discloses antisense oligonucleotides (AONs) for thetargeted editing of RNA, wherein the AONs are characterized by asequence that is complementary to a target RNA sequence (thereinreferred to as the ‘targeting portion’) and by the presence of astem-loop structure (therein referred to as the ‘recruitment portion’).Such oligonucleotides are referred to as ‘axiomer AONs’ or ‘self-loopingAONs’. The recruitment portion acts in recruiting a natural ADAR enzymepresent in the cell to the dsRNA formed by hybridization of the targetsequence with the targeting portion. Due to the recruitment portionthere is no need for conjugated entities or presence of modifiedrecombinant ADAR enzymes. WO 2016/097212 describes the recruitmentportion as being a stem-loop structure mimicking either a naturalsubstrate (e.g. the GluB receptor) or a Z-DNA structure known to berecognized by the dsRNA binding regions of ADAR enzymes. A stem-loopstructure can be an intermolecular stem-loop structure, formed by twoseparate nucleic acid strands, or an intramolecular stem loop structure,formed within a single nucleic acid strand. The stem-loop structure ofthe recruitment portion as described in WO 2016/097212 is anintramolecular stem-loop structure, formed within the AON itself, andable to attract ADAR.

Yet another editing technique which uses oligonucleotides is known asCRISPR/Cas9 system, but this editing complex acts on DNA. The lattermethod suffers from the same drawback as the engineered ADAR systemsdescribed above, as it requires co-delivery to the target cell of theCRISPR/Cas9 enzyme, or an expression construct encoding the same,together with the guide oligonucleotide.

In view of the above, there remains a need for new techniques andcompounds that can utilise endogenous cellular pathways and naturallyavailable ADAR enzymes to specifically edit endogenous nucleic acids inmammalian cells, even in whole organisms, without the problemsassociated with the methods of the prior art.

SUMMARY OF THE INVENTION

The present invention does away with the drawbacks of the methodsaccording to the prior art by providing a targeted approach to RNAediting using, in one embodiment, an antisense oligonucleotide (AON)capable of forming a double stranded complex with a target RNA sequencein a cell, preferably a human cell, for the deamination of a targetadenosine in the target RNA sequence by an ADAR enzyme present in thecell, said AON comprising a Central Triplet of 3 sequential nucleotides,wherein the nucleotide directly opposite the target adenosine is themiddle nucleotide of the Central Triplet, wherein the middle nucleotidein the Central Triplet does not have a 2′-O-methyl modification; andwherein 1, 2 or 3 nucleotides in said Central Triplet comprise a sugarmodification and/or a base modification and/or a phosphodiestermodification (such as phoshoro(di)thioate or amidate). The AON of thepresent invention is preferably in its basic structure a single strandedoligoribonucleotide.

The present invention also relates to the AON according to the inventionfor use in the treatment or prevention of a genetic disorder, preferablyselected from the group consisting of: Cystic fibrosis, Hurler Syndrome,alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer'sdisease, albinism, Amyotrophic lateral sclerosis, Asthma, ß-thalassemia,Cadasil syndrome, Charcot-Marie-Tooth disease, Chronic ObstructivePulmonary Disease (COPD), Distal Spinal Muscular Atrophy (DSMA),Duchenne/Becker muscular dystrophy, Dystrophic Epidermolysis bullosa,Epidermolysis bullosa, Fabry disease, Factor V Leiden associateddisorders, Familial Adenomatous Polyposis, Galactosemia, Gaucher'sDisease, Glucose-6-phosphate dehydrogenase deficiency, Haemophilia,Hereditary Hemachromatosis, Hunter Syndrome, Huntington's disease,Inflammatory Bowel Disease (IBD), Inherited polyagglutination syndrome,Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfansyndrome, Mucopolysaccharidosis, Muscular Dystrophy, Myotonic dystrophytypes I and II, neurofibromatosis, Niemann-Pick disease type A, B and C,NY-eso1 related cancer, Peutz-Jeghers Syndrome, Phenylketonuria, Pompe'sdisease, Primary Ciliary Disease, Prothrombin mutation relateddisorders, such as the Prothrombin G20210A mutation, PulmonaryHypertension, Retinitis Pigmentosa, Sandhoff Disease, Severe CombinedImmune Deficiency Syndrome (SCID), Sickle Cell Anemia, Spinal MuscularAtrophy, Stargardt's Disease, Tay-Sachs Disease, Usher syndrome,X-linked immunodeficiency, Sturge-Weber Syndrome, and cancer.

The invention also relates to a method for the deamination of a specifictarget adenosine present in a target RNA sequence in a cell, said methodcomprising the steps of: providing said cell with an AON according tothe invention; allowing uptake by the cell of said AON; allowingannealing of said AON to the target RNA sequence; allowing a mammalianADAR enzyme comprising a natural dsRNA binding domain as found in thewild type enzyme to deaminate said target adenosine in said target RNAsequence to an inosine; and, optionally identifying the presence of saidinosine in the RNA sequence.

In preferred embodiments of the present invention the target RNAsequence encodes CFTR (e.g. to edit a 1784G>A mutation), CEP290 (e.g. toedit a c.2991+1655A>G mutation), alpha1-antitrypsin (A1AT; e.g. to edita 9989G>A mutation; or a 1096G>A mutation), Guanine Nucleotide BindingProtein (GNAQ; e.g. to edit a 548G>A mutation), or LRRK2 (e.g. to edit aG6055 mutation), or wherein the target RNA is encoded by the IDUA gene(e.g. to edit a c.1205G>A (W402X) mutation).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show the complementarity of an antisenseoligonucleotide (AON; upper strand) to the human mutated SERPINA1 targetRNA sequence (lower strand; SEQ ID NO: 1), with the target adenosine Ain bold. The sequence of the AONs of the ADAR60 series is here shownfrom 3′ to 5′ (in contrast to the direction as it is shown in FIGS.2A-D), with the Central Triplet of the AON underlined. Positions in theCentral Triplet that are modified as described in the provided examplesare indicated by the letters YXZ.

FIGS. 2A-2D show the antisense oligonucleotides (AONs) and senseoligonucleotide (SON) for testing RNA editing and stability assessmentas disclosed herein. Independent sequences have a SEQ ID NO asindicated. Specific YXZ base modifications are mentioned in the thirdcolumn. Lower case nucleotides are RNA and 2′-O-methyl modified. Uppercase nucleotides are RNA, except for bracketed [NNN] nucleotides, whichis DNA. Lower case nucleotides depicted as (nnn) are 2′-fluoro RNAmodified nucleotides. Lower case nucleotides depicted as <nnn> are2′-NH₂ RNA modified nucleotides. Nucleotides depicted as {N} areUnlocked Nucleic Acid (UNA). idT indicates a 3′ inverted T modificationwhich enhances the resistance to degradation and also blocks the3′-terminus of AON from extension during PCR amplification.*=phosphorothioate linkages; =3′-methylenephosphonatelinkages;”=5′-methylenephosphonate linkages; {circumflex over( )}=3′-phosphoroamidate linkages; #=2′-5′ phosphodiester linkages.

FIGS. 3A-3C show the nuclease resistance of AONs, assayed by denaturinggel electrophoresis after incubation in FBS-containing medium (indicatedby RX). Controls were incubated in PBS (CTL). Identities of the AONs(ADAR60-1 through ADAR60-21) are indicated above the panel (abbreviatedto 60-1 through 60-21, see also FIGS. 2A-D). The lower bands (indicatedby an arrow) are degradation products resulting from cleavage of theAONs, while the upper bands are the full-length AONs. The Marker lanescontain Low Molecular Weight Marker, 10-100 nt (Affymetrix), with 14oligonucleotide fragments at 5 nt (10 nt-50 nt) and 10 nt (50 nt-100 nt)increments. Susceptibility to degradation is indicated by the ratio ofthe bands, with a higher prevalence of the lower product suggestinglower stability.

FIG. 4A and FIG. 4B show the complementarity of an AON (upper strand) tothe human mutated IDUA target RNA sequence (lower strand; SEQ ID NO: 2),with the target adenosine A in bold. The sequence of the AONs of theADAR68 series is here shown from 3′ to 5′ (in contrast to the directionas it is shown in FIGS. 2A-D), with the Central Triplet of the AONunderlined. Positions in the Central Triplet that are modified asdescribed in the provided examples are indicated by the letters XXY.

FIG. 5 shows the stability of the oligonucleotides ADAR65-1, -18, -20,-21, and -22 directed against the mutated mouse ldua gene (Hurlermodel), assayed by denaturing gel electrophoresis after incubation inFBS-containing medium. Controls were incubated in PBS. The arrows pointat degradation products. The upper bands are full-length AONs. TheMarker lanes (M) contain Low Molecular Weight Marker, 10-100 nt(Affymetrix), with 14 oligonucleotide fragments at 5 nt (10 nt −50 nt)and 10 nt (50 nt-100 nt) increments. AON susceptibility to degradationover two different time incubations (2 h and 18 h) is indicated by theratio of the bands, with a higher prevalence of the lower productsuggesting reduced stability.

FIG. 6 shows the stability of the additional ADAR65 oligonucleotides,assayed by denaturing gel electrophoresis after incubation for 2 h or 18h in PBS or FBS, similar to FIG. 5 .

FIG. 7 shows the stability of four ADAR93 oligonucleotides, of whichADAR93-2 contains unmodified RNA in the Central Triplet, while the otherthree (ADAR93-6, -8, and -9) have two or three DNA nucleotides in theCentral Triplet instead. The difference of the additional modificationsis as shown in FIGS. 2A-D. The stability was assayed by denaturing gelelectrophoresis after incubation for 2 h, 24 h or 3 days in PBS,DMEM+15% FBS, Single Donor Human Cerebrospinal Fluid (CSF) and MixedGender Human Liver Lysosomes (Lyso).

FIGS. 8A-8D show the Sanger sequencing analysis of PCR fragmentsgenerated by RT-PCR after in vitro editing assay using HEK293 celllysate with overexpressed ADAR2 and a SERPINA1 mutant ssRNA target.ADAR60-1 (FIG. 8A) and ADAR60-15 (FIG. 8B) were used, see FIGS. 2A-D.Negative controls were an identical assay with ADAR60-15 but withoutcell lysate (FIG. 8C), and an identical assay using the HEK293 celllysate (with ADAR2 overexpression) in the absence of AON (FIG. 8D). Thesequence above all panels is SEQ ID NO:35.

FIG. 9 shows the sequencing results of the RT-PCR amplified GFP RNA fromcells that stably express the GFP W57X construct and that weretransfected with ADAR2 and separately with two oligonucleotides(ADAR59-2 and ADAR59-10), wherein the adenine in the Central Triplet ofADAR59-10 was a 2-aminopurine. ADAR59-2 does not have that modification,see FIGS. 2A-D. It shows the enhancing effect of this particularmodification on RNA editing efficiency. The sequence above both panelsis SEQ ID NO:36.

FIG. 10A and FIG. 10B show the sequencing results of a PCR productgenerated via cDNA from RNA isolated from mouse HEPA1-6 cells (FIG. 10A)and CMT64 cells (FIG. 10B) that were either not-transfected (NT, leftpanels) or transfected with a non-targeting control oligonucleotide(NTO, middle panels) or with ADAR94-1 (right panels) to edit the stopcodon of the mouse Snrpa mRNA. RNA editing that is clearly abovebackground levels is observed at the position indicated by the arrow.The sequence above all three panels in FIG. 10A and FIG. 10B is SEQ IDNO:37.

FIG. 11 shows the sequencing results of a PCR product generated via cDNAfrom RNA isolated from mouse HEPA1-6 cells that were transfected withADAR94-1 alone or ADAR94-1 in combination with SON2 as described in FIG.10A and FIG. 10B. The sequence above both panels is SEQ ID NO:38.

FIG. 12 shows the sequencing results of a PCR product generated via cDNAfrom RNA isolated from primary mouse lung cells that were either nottransfected (NT, left panel), transfected with a control oligonucleotide(ADAR87-1) annealed to a protecting sense oligonucleotide SON2 (middlepanel), or with an oligonucleotide targeting the mouse Snrpa RNA(ADAR94-1) annealed to SON2 (right panel). A clear increase in G signalis seen after transfection with ADAR94-1+SON2 (position indicated by anarrow), which shows that highly specific and significant RNA editing canbe achieved with endogenous ADAR on an endogenous target in ex vivoprimary cells. The sequence above all three panels is SEQ ID NO:37,identical to FIG. 10A and FIG. 10B.

FIG. 13 shows the editing ability of AONs as a percentage of edited mRNAin total target RNA measured by ddPCR. NT: Non-treated.

FIG. 14 shows the enzymatic activity (as relative fluorescence unitsnormalized to total protein concentration) measured in anα-L-iduronidase assay which is an indication of the presence of a wtldua mRNA that is a result of RNA editing upon transfection withRNA-editing AONs. Mouse embryonic fibroblasts carrying the mutated lduagene were transfected with an expression plasmid also expressing themouse mutated ldua gene, and subsequently transfected with the AONs asdepicted. Average activity and standard deviation from two duplicatemeasurements is shown for each AON. NT: Non-transfected with AON.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, WO 2016/097212 discloses AONs for the targetedediting of RNA, wherein the AONs are characterized by a ‘targetingportion’ and a ‘recruitment portion’. WO 2016/097212 describes therecruitment portion as being a stem-loop structure mimicking either anatural substrate (e.g. the GluB receptor) or a Z-DNA structure known tobe recognized by the dsRNA binding regions of ADAR enzymes. Thestem-loop structure of the recruitment portion as described in WO2016/097212 is an intramolecular stem-loop structure, formed within theAON itself, and able to attract ADAR. There are potential disadvantagesin having AONs that are very long because of such structures, such asproblems with manufacturing, stability, side-effects and they are moreexpensive to produce than shorter versions.

The AONs of the present invention do not comprise a recruitment portionas described in WO 2016/097212. The AONs of the present invention do notcomprise a portion that is capable of forming an intramolecularstem-loop structure. The AONs of the present invention are shorter,which makes them cheaper to produce, easier to use and easier tomanufacture. Furthermore, they do not have the disadvantage ofpotentially sequestering ADAR enzymes from their normal function in thecell. Unexpectedly, it was found that AONs that are complementary to atarget RNA for deaminating a target adenosine present in a target RNAsequence to which the AON is complementary, but—importantly—lack arecruitment portion as described above, appeared still capable ofharnessing ADAR enzymes present in the cell to edit the targetadenosine. In a preferred aspect the AON of the present inventioncomprises a mismatch at the position of the target adenosine, whereinthe opposite nucleotide is a cytidine. Also when a uridine is oppositethe target adenosine (which would in fact not be a mismatch), the AON iscapable of bringing about deamination of the target adenosine.PCT/EP2017/065467 describes that additional mismatches (resulting inso-called ‘bulges’ in the formed dsRNA, caused by nucleotides in the AONthat do not form perfect base pairs with the target RNA according to theWatson-Crick base pairing rules) are tolerable, in some casespreferable, but not essential for specific targeted editing of thetarget RNA sequence. The number of bulges in the AON (when it hybridisesto its RNA target sequence) may be one (usually the bulge formed at thetarget adenosine position) or more, depending on the length of the AON.The additional bulge-inducing mismatches may be upstream as well asdownstream of the target adenosine. The bulges may be single-mismatchbulges (caused by one mismatching base pair) or multi-mismatch bulges(caused by more than one consecutive mismatching base pairs, preferablytwo or three consecutive mismatching base pairs).

The AONs according to the present invention have certain advantages overthe oligonucleotides described in WO 2016/097212 and PCT/EP2017/065467,in that there is no need for hairpin or stem-loop structures, whichallow the AONs of the present invention to be (considerably) shorter.The oligonucleotides described in WO 2016/097212 bear the potential riskof sequestering ADAR enzyme present in the cell. By sequestering in thiscontext it is meant that a natural ADAR protein may bind to theoligonucleotides even in the absence of the formation of a dsRNA complexbetween the targeting portion of the oligonucleotide and the target RNA.This direct binding of ADAR to the oligonucleotides due to the presenceof an intramolecular stem-loop structure, in the absence of target RNAsequences does not take place when using the AONs of the presentinvention, which do not comprise a portion that is capable of forming anintramolecular stem-loop structure. There are many instances where thepresence of the hairpin and/or (stem-) loop structures is preferablyavoided.

It is an important aspect of the invention that the AON comprises one ormore nucleotides with one or more sugar modifications. Thereby, a singlenucleotide of the AON can have one, or more than one sugar modification.Within the AON, one or more nucleotide(s) can have such sugarmodification(s).

It is an important aspect of the invention that the nucleotide withinthe AON of the present invention that is opposite to the nucleotide thatneeds to be edited does not contain a 2′-O-methyl modification (hereinand elsewhere often referred to as a 2′-OMe group, as 2′-O-methylation,or as a 2′-O-methyl group). It is preferred that the nucleotides thatare directly 3′ and 5′ of this nucleotide (the ‘neighbouringnucleotides’ in the Central Triplet) also lack such a chemicalmodification, although it is believed that it is tolerated that one orboth of the neighbouring nucleotides may contain a 2′-O-alkyl group(such as a 2′-O-methyl group). Either one, or both neighbouringnucleotides or all three nucleotides of the Central Triplet may be 2′-OHor a compatible substitution (as defined herein).

Another important aspect of the AON of the present invention is that itdoes not have a portion (which is not complementary to the targetsequence or the region that comprises the target adenosine) that allowsthe AON in itself to fold into an intramolecular hairpin or other typeof (stem-) loop structure (herein also referred to as “auto-looping” or“self-looping”) under physiological conditions, and which maypotentially act as a structure that sequesters ADAR. In a preferredaspect, the single stranded AON of the present invention is completelycomplementary with the target RNA, although it may optionally mismatchat several positions, especially the position at the target adenosine.

Preferred AONs of the present invention do not include a 5′-terminalO6-benzylguanosine or a 5′-terminal amino modification, and are notcovalently linked to a SNAP-tag domain (an engineeredO6-alkylguanosine-DNA-alkyl transferase) in contrast to Vogel et al.(2014). The SNAP-tag domain is derived from the human DNA repair proteinO6-alkylguanosine-DNA-alkyl transferase (AGT) and can be covalentlylabelled in living cells using O6-benzylguanosine derivatives. Vogel etal. (2014) discloses guide RNAs with a total length of either 20 or 17nucleotides, wherein the first three nucleotides at the 5′ end do notbind to the target RNA sequence, but link the guide RNA to the SNAP-tagdomain. The portion of the guide RNA which binds to the target RNAsequence is therefore either 14 or 17 nucleotides in length. Guide RNAs,of the same lengths, with a 5′-terminal amino modification in place ofthe 5′-terminal O6-benzylguanosine modification are also disclosed inVogel et al. (2014), however only very little, or no deamination or thetarget RNA sequence was detected. In one embodiment, the AON of thepresent invention comprises 0, 1, 2 or 3 mismatches with the target RNAsequence, wherein a single mismatch may comprise multiple sequentialnucleotides.

Similarly, a preferred AON of the present invention does not include aboxB RNA hairpin sequence, in contrast to Montiel-Gonzalez et al (2013).The boxB RNA hairpin sequence used in Montiel-Gonzalez et al. (2013) isa short stretch of RNA of 17 nucleotides that is recognized by thebacteriophage lambda N-protein. Transcription of downstream genes in theearly operons of bacteriophage requires a promoter-proximal elementknown as nut. This site acts in cis in the form of RNA to assemble atranscription anti-termination complex which is composed of abacteriophage lambda N protein and host factors. The nut-site RNAcontains a small stem-loop structure called boxB. The boxB RNA hairpinsequence is known in the art as an interrupted palindrome with thepotential to form a hairpin (stem-loop) structure. Its sequence variesamong relatives of bacteriophage lambda which encode distinct genomespecific N homologues.

Neither Vogel et al. (2014), nor Montiel-Gonzalez et al (2013) use amammalian ADAR enzyme present in the cell, wherein the ADAR enzymecomprises its natural dsRNA binding domain as found in the wild-typeenzyme. Vogel et al. (2014) uses a genetically engineered fusion proteincomprising the adenosine deaminase domain of ADAR1 or 2 fused to aSNAP-tag domain and Montiel-Gonzalez et al uses a genetically engineeredfusion protein comprising the adenosine deaminase domain of the hADAR2protein, fused to the boxB recognition domain of bacteriophage lambda Nprotein. In contrast to the prior art, the AONs of the present inventionuse a mammalian ADAR enzyme present in the cell, wherein the ADAR enzymecomprises its natural dsRNA binding domain as found in the wild typeenzyme. There is therefore no need to incorporate a boxB RNA hairpinsequence, a 5′-terminal 06-benzylguanosine, a 5′-terminal aminomodification, or a SNAP-tag domain into the AON of the presentinvention, to allow recruitment of ADAR. The AONs according to thepresent invention therefore have certain advantages over theoligonucleotides described in Vogel et al. (2014) and Montiel-Gonzalezet al (2013). The AONs according to the present invention can utiliseendogenous cellular pathways and naturally available ADAR enzymes tospecifically edit a target adenosine in a target RNA sequence. In oneembodiment, an AON of the invention is not covalently linked to a human06-alkylguanosine-DNA-alkyl transferase. Preferably, an AON of theinvention is not covalently linked to a polypeptide. In another aspectof the AON of the present invention, the AON does not have a 5′ cap. Ineukaryotes, the 5′ cap consists of a guanosine nucleotide connected tothe RNA via a 5′ to 5′ triphosphate linkage. This guanosine ismethylated on the 7 position and is referred to as a 7-methylguanosine.

AONs of the invention are capable of deamination of a specific targetadenosine nucleotide in a target RNA sequence. Thus, ideally only oneadenosine is deaminated. Alternatively 1, 2, or 3 adenosine nucleotidesare deaminated, for instance when target adenosines are in closeproximity of each other. It would then require the presence of two ormore ‘Central Triplets’ in a single AON, and depending of the distances,such may be used. However, if the distance is too big to cover multipletarget adenosines by a single AON, the AON preferably comprises only oneCentral Triplet opposite the specified and single target adenosine.

Taking the features of the AONs of the present invention together: thereis no need for modified recombinant ADAR expression; there is no needfor conjugated entities attached to the AON; or the presence of longrecruitment portions that are not complementary to the target RNAsequence. Besides that, the AON of the present invention does allow forthe specific deamination of a target adenosine present in the target RNAsequence to an inosine by a natural ADAR enzyme comprising a naturaldsRNA binding domain as found in the wild-type enzyme, without the riskof promiscuous editing elsewhere in the dsRNA complex.

The recruitment of cytidine deaminase to a target site works in the sameway as for the adenosine deaminases hADAR1 and hADAR2. However, cytidinedeaminases have different binding requirements and recognize differentstructures in their target RNA sequences that determine editing of thecytidine. One particularly well studied cytidine deaminase is humanApobec1. The general principle of RNA editing using an oligonucleotideconstruct to target an editing site and to recruit a resident, naturallypresent, editing entity remains the same for cytidine deaminases, and ispart of the invention disclosed and claimed herein.

Analysis of natural targets of ADAR enzymes has indicated that thesegenerally include mismatches between the two strands that form the RNAhelix edited by ADAR1 or 2. It has been suggested that these mismatchesenhance the specificity of the editing reaction (Stefl et al. 2006.Structure 14(2):345-355; Tian et al. 2011. Nucleic Acids Res39(13):5669-5681). Characterization of optimal patterns ofpaired/mismatched nucleotides between the AONs and the target RNA alsoappears crucial for development of efficient ADAR-based AON therapy.

Another improved feature of the AONs of the present invention is the useof specific nucleotide modifications at predefined spots to ensurestability as well as proper ADAR binding and activity. These changes mayvary and may include modifications in the backbone of the AON, in thesugar moiety of the nucleotides as well as in the nucleobases or thephosphodiester linkages. They may also be variably distributedthroughout the sequence of the AON. Specific modifications may be neededto support interactions of different amino acid residues within theRNA-binding domains of ADAR enzymes, as well as those in the deaminasedomain. For example, phosphorothioate linkages between nucleotides or2′-O-methyl modifications may be tolerated in some parts of the AON,while in other parts they should be avoided so as not to disrupt crucialinteractions of the enzyme with the phosphate and 2′-OH groups. Part ofthese design rules are guided by the published structures of ADAR2,while others have to be defined empirically. Different preferences mayexist for ADAR1 and ADAR2. The modifications should also be selectedsuch that they prevent degradation of the AONs.

Specific nucleotide modifications may also be necessary to enhance theediting activity on substrate RNAs where the target sequence is notoptimal for ADAR editing. Previous work has established that certainsequence contexts are more amenable to editing. For example, the targetsequence 5′-UAG-3′ (with the target A in the middle) contains the mostpreferred nearest-neighbor nucleotides for ADAR2, whereas a 5′-CAA-3′target sequence is disfavored (Schneider et al. 2014. Nucleic Acids Res42(10):e87). The recent structural analysis of ADAR2 deaminase domainhints at the possibility of enhancing editing by careful selection ofthe nucleotides that are opposite to the target trinucleotide. Forexample, the 5′-CAA-3′ target sequence, paired to a 3′-GCU-5′ sequenceon the opposing strand (with the A-C mismatch formed in the middle), isdisfavored because the guanosine base sterically clashes with an aminoacid side chain of ADAR2. However, here it is postulated that a smallernucleobase, such as inosine, could potentially fit better into thisposition without causing steric clashes, while still retaining thebase-pairing potential to the opposing cytosine. Modifications thatcould enhance activity of suboptimal sequences include the use ofbackbone modifications that increase the flexibility of the AON or,conversely, force it into a conformation that favors editing.

Definitions of Terms as Used Herein

The ‘Central Triplet’ as used and defined herein are the threenucleotides opposite the target adenosine in the target RNA, wherein themiddle nucleotide in the Central Triplet is directly opposite the targetadenosine. The Central Triplet does not have to be in the middle (in thecentre) of the AON, it may be located more to the 3′ as well as to the5′ end of the AON, whatever is preferred for a certain target. Centralin this aspect has therefore more the meaning of the triplet that is inthe centre of catalytic activity when it comes to chemical modificationsand targeting the target adenosine. It should also be noted that theAONs are sometimes depicted from 3′ to 5′, especially when the targetsequence is shown from 5′ to 3′. However, whenever herein the order ofnucleotides within the AON is discussed it is always from 5′ to 3′ ofthe AON. For example, the first nucleotide of the Central Triplet inADAR60-1 is the U of the 5′-UCG-3′ triplet. The position can also beexpressed in terms of a particular nucleotide within the AON while stilladhering to the 5′ to 3′ directionality, in which case other nucleotides5′ of the said nucleotide are marked as negative positions and those 3′of it as positive positions. For example, the C in the Central tripletis the nucleotide (at the 0 position) opposite the targeted adenosineand the U would in this case be the −1 nucleotide and the G would thenbe the +1 nucleotide, etc.

As outlined herein, and in most examples of AONs disclosed herein, thenucleotides outside the Central triplet are 2′-O-methyl modified.However, this is not a requirement of the AONs of the present invention.The use of 2′-O-methylation in those nucleotides assures a properstability of those parts of the AON, but other modifications may beapplied as well, such as 2′-O-methoxyethyl (2′-O-MOE) modifications.

The terms ‘adenine’, ‘guanine’, ‘cytosine’, ‘thymine’, ‘uracil’ and‘hypoxanthine’ (the nucleobase in inosine) as used herein refer to thenucleobases as such.

The terms ‘adenosine’, ‘guanosine’, ‘cytidine’, ‘thymidine’, ‘uridine’and ‘inosine’, refer to the nucleobases linked to the (deoxy)ribosylsugar.

The term ‘nucleoside’ refers to the nucleobase linked to the(deoxy)ribosyl sugar.

The term ‘nucleotide’ refers to the respectivenucleobase-(deoxy)ribosyl-phospholinker, as well as any chemicalmodifications of the ribose moiety or the phospho group. Thus the termwould include a nucleotide including a locked ribosyl moiety (comprisinga 2′-4′ bridge, comprising a methylene group or any other group, wellknown in the art), a nucleotide including a linker comprising aphosphodiester, phosphotriester, phosphoro(di)thioate,methylphosphonates, phosphoramidate linkers, and the like.

Sometimes the terms adenosine and adenine, guanosine and guanine,cytosine and cytidine, uracil and uridine, thymine and thymidine,inosine and hypo-xanthine, are used interchangeably to refer to thecorresponding nucleobase, nucleoside or nucleotide.

Sometimes the terms nucleobase, nucleoside and nucleotide are usedinterchangeably, unless the context clearly requires differently.

Whenever reference is made to an ‘oligonucleotide’, botholigoribonucleotides and deoxyoligoribonucleotides are meant unless thecontext dictates otherwise. Whenever reference is made to an‘oligoribonucleotide’ it may comprise the bases A, G, C, U or I.Whenever reference is made to a ‘deoxyoligoribonucleotide’ it maycomprise the bases A, G, C, T or I. In a preferred aspect, the AON ofthe present invention is an oligoribonucleotide that may comprisechemical modifications.

Whenever reference is made to nucleotides in the oligonucleotideconstruct, such as cytosine, 5-methylcytosine, 5-hydroxymethylcytosine,Pyrrolocytidine, and β-D-Glucosyl-5-hydroxymethylcytosine are included;when reference is made to adenine, 2-aminopurine, 2,6-diaminopurine,3-deazaadenosine, 7-deazaadenosine, 8-azidoadenosine, 8-methyladenosine,7-aminomethyl-7-deazaguanosine, 7-deazaguanosine, N6-Methyladenine and7-methyladenine are included; when reference is made to uracil,5-methoxyuracil, 5-methyluracil, dihydrouracil, pseudouracil, andthienouracil, dihydrouracil, 4-thiouracil and 5-hydroxymethyluracil areincluded; when reference is made to guanosine, 7-methylguanosine,8-aza-7-deazaguanosine, thienoguanosine and 1-methylguanosine areincluded.

Whenever reference is made to nucleosides or nucleotides, ribofuranosederivatives, such as 2′-deoxy, 2′-hydroxy, 2-fluororibose and2′-O-substituted variants, such as 2′-O-methyl, are included, as well asother modifications, including 2′-4′ bridged variants.

Whenever reference is made to oligonucleotides, linkages between twomono-nucleotides may be phosphodiester linkages as well as modificationsthereof, including, phosphodiester, phosphotriester,phosphoro(di)thioate, methylphosphonate, phosphor-amidate linkers, andthe like.

The term ‘comprising’ encompasses ‘including’ as well as ‘consisting’,e.g. a composition ‘comprising X’ may consist exclusively of X or mayinclude something additional, e.g. X+Y.

The term ‘about’ in relation to a numerical value x is optional andmeans, e.g. x±10%.

The word ‘substantially’ does not exclude ‘completely’, e.g. acomposition which is ‘substantially free from Y’ may be completely freefrom Y. Where relevant, the word ‘substantially’ may be omitted from thedefinition of the invention.

The term ‘downstream’ in relation to a nucleic acid sequence meansfurther along the sequence in the 3′ direction; the term ‘upstream’means the converse. Thus in any sequence encoding a polypeptide, thestart codon is upstream of the stop codon in the sense strand, but isdownstream of the stop codon in the antisense strand.

References to ‘hybridisation’ typically refer to specific hybridisation,and exclude non-specific hybridisation. Specific hybridisation can occurunder experimental conditions chosen, using techniques well known in theart, to ensure that the majority of stable interactions between probeand target are where the probe and target have at least 70%, preferablyat least 80%, more preferably at least 90% sequence identity.

The term ‘mismatch’ is used herein to refer to opposing nucleotides in adouble stranded RNA complex which do not form perfect base pairsaccording to the Watson-Crick base pairing rules. Mismatch base pairsare G-A, C-A, U-C, A-A, G-G, C-C, U-U base pairs. In some embodimentsAONs of the present invention comprise 0, 1, 2 or 3 mismatches, whereina single mismatch may comprise several sequential nucleotides. Wobblebase pairs are: G-U, I-U, I-A, and I-C base pairs.

An AON according to the present invention may be chemically modifiedalmost in its entirety, for example by providing all nucleotides with a2′-O-methylated sugar moiety (2′-OMe). However, the nucleotide oppositethe target adenosine does not comprise the 2′-O-methyl modification, andin yet a further preferred aspect, at least one of the two neighbouringnucleotides flanking the nucleotide opposing the target adenosine doesnot comprise a 2′-O-methyl modification. Complete modification, whereinall nucleotides in the AON holds a 2′-O-methyl modification (includingthe Central Triplet) results in a non-functional oligonucleotide as faras RNA editing goes, presumably because it hinders the ADAR activity atthe targeted position. In general, an adenosine in a target RNA can beprotected from editing by providing an opposing nucleotide with a2′-O-methyl group, or by providing a guanosine or adenosine as opposingbase, as these two nucleobases are also able to reduce editing of theopposing adenosine.

Various chemistries and modification are known in the field ofoligonucleotides that can be readily used in accordance with theinvention. The regular internucleosidic linkages between the nucleotidesmay be altered by mono- or di-thioation of the phosphodiester bonds toyield phosphorothioate esters or phosphorodithioate esters,respectively. Other modifications of the internucleosidic linkages arepossible, including amidation and peptide linkers. In a preferred aspectthe AONs of the present invention have one, two, three, four or morephosphorothioate linkages between the most terminal nucleotides of theAON (hence, preferably at both the 5′ and 3′ end), which means that inthe case of four phosphorothioate linkages, the ultimate 5 nucleotidesare linked accordingly. It will be understood by the skilled person thatthe number of such linkages may vary on each end, depending on thetarget sequence, or based on other aspects, such as toxicity.

The ribose sugar may be modified by substitution of the 2′-0 moiety witha lower alkyl (C1-4, such as 2′-O-methyl), alkenyl (C2-4), alkynyl(C2-4), methoxyethyl (2′-O-MOE), —H (as in DNA) or other substituent.Preferred substituents of the 2′-OH group are a methyl, methoxyethyl or3,3′-dimethylallyl group. The latter is known for its property toinhibit nuclease sensitivity due to its bulkiness, while improvingefficiency of hybridization (Angus & Sproat. 1993. FEBS Vol. 325, no. 1,2, 123-7). Alternatively, locked nucleic acid sequences (LNAs),comprising a 2′-4′ intramolecular bridge (usually a methylene bridgebetween the 2′ oxygen and 4′ carbon) linkage inside the ribose ring, maybe applied. Purine nucleobases and/or pyrimidine nucleobases may bemodified to alter their properties, for example by amination ordeamination of the heterocyclic rings. The exact chemistries and formatsmay depend from oligonucleotide construct to oligonucleotide constructand from application to application, and may be worked out in accordancewith the wishes and preferences of those of skill in the art. It isbelieved in the art that 4 or more consecutive DNA nucleotides (4consecutive deoxyriboses) in an oligonucleotide create so-called gapmersthat—when annealed to their RNA cognate sequences—induce cleavage of thetarget RNA by RNaseH. According to the present invention, RNaseHcleavage of the target RNA is generally to be avoided as much aspossible.

The AON according to the invention should normally be longer than 10nucleotides, preferably more than 11, 12, 13, 14, 15, 16, still morepreferably more than 17 nucleotides. In one embodiment the AON accordingto the invention is longer than 20 nucleotides. The oligonucleotideaccording to the invention is preferably shorter than 100 nucleotides,still more preferably shorter than 60 nucleotides. In one embodiment theAON according to the invention is shorter than 50 nucleotides. In apreferred aspect, the oligonucleotide according to the inventioncomprises 18 to 70 nucleotides, more preferably comprises 18 to 60nucleotides, and even more preferably comprises 18 to 50 nucleotides.Hence, in a most preferred aspect, the oligonucleotide of the presentinvention comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 or 50 nucleotides.

It is known in the art, that RNA editing entities (such as human ADARenzymes) edit dsRNA structures with varying specificity, depending on anumber of factors. One important factor is the degree of complementarityof the two strands making up the dsRNA sequence. Perfect complementarityof the two strands usually causes the catalytic domain of hADAR todeaminate adenosines in a non-discriminative manner, reacting more orless with any adenosine it encounters. The specificity of hADAR1 and 2can be increased by ensuring a number of mismatches in the dsRNA, whichpresumably help to position the dsRNA binding domains in a way that hasnot been clearly defined yet. Additionally, the deamination reactionitself can be enhanced by providing an AON that comprises a mismatchopposite the adenosine to be edited. The mismatch is preferably createdby providing a targeting portion having a cytidine opposite theadenosine to be edited. As an alternative, also uridines may be usedopposite the adenosine, which, understandably, will not result in a‘mismatch’ because U and A pair. Upon deamination of the adenosine inthe target strand, the target strand will obtain an inosine which, formost biochemical processes, is “read” by the cell's biochemicalmachinery as a G. Hence, after A to I conversion, the mismatch has beenresolved, because I is perfectly capable of base pairing with theopposite C in the targeting portion of the oligonucleotide constructaccording to the invention. After the mismatch has been resolved due toediting, the substrate is released and the oligonucleotideconstruct-editing entity complex is released from the target RNAsequence, which then becomes available for downstream biochemicalprocesses, such as splicing and translation.

The desired level of specificity of editing the target RNA sequence maydepend from application to application. Following the instructions inthe present patent application, those of skill in the art will becapable of designing the complementary portion of the oligonucleotideaccording to their needs, and, with some trial and error, obtain thedesired result.

The oligonucleotide of the invention will usually comprise the normalnucleotides A, G, U and C, but may also include inosine (I), for exampleinstead of one or more G nucleotides.

To prevent undesired editing of adenosines in the target RNA sequence inthe region of overlap with the oligonucleotide construct, theoligonucleotide may be chemically modified. It has been shown in theart, that 2′-O-methylation of the ribosyl-moiety of a nucleosideopposite an adenosine in the target RNA sequence dramatically reducesdeamination of that adenosine by ADAR (Vogel et al. 2014). This is asevere drawback for using such AONs in therapeutic settings and it is apurpose of the present invention to solve, at least in part, thatproblem of having unmodified RNA nucleotides in an AON, which makes itprone to degradation.

RNA editing molecules in the cell will usually be proteinaceous innature, such as the ADAR enzymes found in metazoans, including mammals.Preferably, the editing entity is an enzyme, more preferably anadenosine deaminase or a cytidine deaminase, still more preferably anadenosine deaminase. The ones of most interest are the human ADARs,hADAR1 and hADAR2, including any isoforms thereof such as hADAR1 p110and p150. RNA editing enzymes known in the art, for whicholigonucleotide constructs according to the invention may convenientlybe designed, include the adenosine deaminases acting on RNA (ADARs),such as hADAR1 and hADAR2 in humans or human cells and cytidinedeaminases. Human ADAR3 (hADAR3) has been described in the prior art,but reportedly has no deaminase activity. It is known that hADAR1 existsin two isoforms; a long 150 kDa interferon inducible version and ashorter, 100 kDa version, that is produced through alternative splicingfrom a common pre-mRNA. Consequently, the level of the 150 kDa isoformpresent in the cell may be influenced by interferon, particularlyinterferon-gamma (IFN-gamma). hADAR1 is also inducible by TNF-alpha.This provides an opportunity to develop combination therapy, wherebyinterferon-gamma or TNF-alpha and oligonucleotides according to theinvention are administered to a patient either as a combination product,or as separate products, either simultaneously or subsequently, in anyorder. Certain disease conditions may already coincide with increasedIFN-gamma or TNF-alpha levels in certain tissues of a patient, creatingfurther opportunities to make editing more specific for diseasedtissues.

Examples of chemical modifications in the AONs of the present inventionare modifications of the sugar moiety, including by cross-linkingsubstituents within the sugar (ribose) moiety (e.g. as in locked nucleicacids: LNA), by substitution of the 2′-O atom with alkyl (e.g.2′-O-methyl), alkynyl (2′-O-alkynyl), alkenyl (2′-O-alkenyl),alkoxyalkyl (e.g. methoxyethyl: 2′-O-MOE) groups, having a length asspecified above, and the like. In addition, the phosphodiester group ofthe backbone may be modified by thioation, dithioation, amidation andthe like to yield phosphorothioate, phosphorodithioate, phosphoramidate,etc., internucleosidic linkages. The internucleotidic linkages may bereplaced in full or in part by peptidic linkages to yield inpeptidonucleic acid sequences and the like. Alternatively, or inaddition, the nucleobases may be modified by (de)amination, to yieldinosine or 2′6′-diaminopurines and the like. A further modification maybe methylation of the C5 in the cytidine moiety of the nucleotide, toreduce potential immunogenic properties known to be associated with CpGsequences.

In case the dsRNA complex recruits ADAR enzymes to deaminate an A to anI in the target RNA sequence, the base-pair, mismatch, bulge or wobblebetween the adenosine to be edited and the opposite nucleotide maycomprise an adenosine, a guanosine, an uridine or a cytidine residue,but preferably a cytidine residue. Except for the potential mismatchopposite the editing site (when no uridine is applied), the remainingportion of the AON may be perfectly complementary to the target RNA.However, as shown herein, in certain aspects the invention relates toAONs that comprise a limited number of imperfect matches. It will beunderstood by a person having ordinary skill in the art that the extentto which the editing entities inside the cell are redirected to othertarget sites may be regulated by varying the affinity of theoligonucleotides according to the invention for the recognition domainof the editing molecule. The exact modification may be determinedthrough some trial and error and/or through computational methods basedon structural interactions between the oligonucleotide and therecognition domain of the editing molecule.

In addition, or alternatively, the degree of recruiting and redirectingthe editing entity resident in the cell may be regulated by the dosingand the dosing regimen of the oligonucleotide. This is something to bedetermined by the experimenter (in vitro) or the clinician, usually inphase I and/or II clinical trials. The invention concerns themodification of target RNA sequences in eukaryotic, preferably metazoan,more preferably mammalian cells. In principle the invention can be usedwith cells from any mammalian species, but it is preferably used with ahuman cell.

The invention can be used with cells from any organ e.g. skin, lung,heart, kidney, liver, pancreas, gut, muscle, gland, eye, brain, bloodand the like. The invention is particularly suitable for modifyingsequences in cells, tissues or organs implicated in a diseased state ofa (human) subject. Such cells include but are not limited to epithelialcells of the lung or the gastrointestinal tract, cells of thereproductive organs, muscle cells, cells of the eye, cells of the skin,cells from tissues and organs such as liver, kidney, pancreas, immunecells, cancerous cells, gland cells, brain cells, and the like. Theinvention can also be used with mammalian cells which are not naturallypresent in an organism e.g. with a cell line or with an embryonic stem(ES) cell. The invention can be used with various types of stem cell,including pluripotent stem cells, totipotent stem cells, embryonic stemcells, induced pluripotent stem cells, etc. The cell can be located invitro or in vivo. One advantage of the invention is that it can be usedwith cells in situ in a living organism, but it can also be used withcells in culture. In some embodiments cells are treated ex vivo and arethen introduced into a living organism (e.g. re-introduced into anorganism from whom they were originally derived). The invention can alsobe used to edit target RNA sequences in cells within a so-calledorganoid. Organoids can be thought of as three-dimensional invitro-derived tissues but are driven using specific conditions togenerate individual, isolated tissues (e.g. see Lancaster & Knoblich,Science 2014, vol. 345 no. 6194 1247125). In a therapeutic setting theyare useful because they can be derived in vitro from a patient's cells,and the organoids can then be re-introduced to the patient as autologousmaterial which is less likely to be rejected than a normal transplant.Thus, according to another preferred embodiment, the invention may bepractised on organoids grown from tissue samples taken from a patient(e.g. from their gastrointestinal tract; see Sala et al. J Surg Res.2009; 156(2):205-12, and also Sato et al. Gastroenterology 2011;141:1762-72); upon RNA editing in accordance with the invention, theorganoids, or stem cells residing within the organoids, may be used totransplant back into the patient to ameliorate organ function.

The cell to be treated will generally have a genetic mutation. Themutation may be heterozygous or homozygous. The invention will typicallybe used to modify point mutations, such as N to A mutations, wherein Nmay be G, C, U (on the DNA level T), preferably G to A mutations, or Nto C mutations, wherein N may be A, G, U (on the DNA level T),preferably U to C mutations. Genes containing mutations of particularinterest are discussed below. In some embodiments, however, theinvention is used in the opposite way by introducing adisease-associated mutation into a cell line or an animal, in order toprovide a useful research tool for the disease in question. As anexample of creating a disease model, one can provide an oligonucleotidesequence that provides for the recruitment of editing activity in ahuman cell to create a mutation in the CEP290 gene, creating a crypticsplice site that forms the basis for a form of Leber's CongenitalAmaurosis, the most common form of congenital child blindness. It canalso be envisioned that non-mutated sequences are targeted for RNAediting, for instance to alter the splicing of a particular target RNA,such that a mutant version of that target gene is translated in anotherfashion, thereby relieving disease. The person skilled in the art isaware of all types of possibilities to modify target RNA for a widevariety of purposes.

A mutation to be reverted through RNA editing may have arisen on thelevel of the chromosome or some other form of DNA, such as mitochondrialDNA, or RNA, including pre-mRNA, ribosomal RNA or mitochondrial RNA. Achange to be made may be in a target RNA of a pathogen, including fungi,yeasts, parasites, kinetoplastids, bacteria, phages, viruses etc, withwhich the cell or subject has been infected. Subsequently, the editingmay take place on the RNA level on a target sequence inside such cell,subject or pathogen. Certain pathogens, such as viruses, release theirnucleic acid, DNA or RNA into the cell of the infected host (cell).Other pathogens reside or circulate in the infected host. Theoligonucleotide constructs of the invention may be used to edit targetRNA sequences residing in a cell of the infected eukaryotic host, or toedit a RNA sequence inside the cell of a pathogen residing orcirculating in the eukaryotic host, as long as the cells where theediting is to take place contain an editing entity compatible with theoligonucleotide construct administered thereto.

Without wishing to be bound by theory, the RNA editing through hADAR1and hADAR2 is thought to take place on primary transcripts in thenucleus, during transcription or splicing, or in the cytoplasm, wheree.g. mature mRNA, miRNA or ncRNA can be edited. Different isoforms ofthe editing enzymes are known to localize differentially, e.g. withhADAR1 p110 found mostly in the nucleus, and hADAR1 p150 in thecytoplasm. The RNA editing by cytidine deaminases is thought to takeplace on the mRNA level. Editing of mitochondrial RNA codons ornon-coding sequences in mature mRNAs is not excluded.

The invention is used to make a change in a target RNA sequence in aeukaryotic cell through the use of an oligonucleotide that is capable oftargeting a site to be edited and recruiting RNA editing entitiesresident in the cell to bring about the editing reaction(s). Preferredediting reactions are adenosine deaminations and cytidine deaminations,converting adenosines into inosines and cytidines into uridines,respectively. The changes may be in 5′ or 3′ untranslated regions of atarget RNA, in (cryptic) splice sites, in exons (changing amino acids inprotein translated from the target RNA, codon usage or splicingbehaviour by changing exonic splicing silencers or enhancers, byintroducing or removing start or stop codons), in introns (changingsplicing by altering intronic splicing silencers or intronic splicingenhancers, branch points) and in general in any region affecting RNAstability, structure or functioning. The target RNA sequence maycomprise a mutation that one may wish to correct or alter, such as apoint mutation (a transition or a transversion). Alternatively, thetarget RNA sequence is deliberately mutated to create an alteredphenotype (or genotype, in case of RNA based organisms, such as RNAviruses), where there was no mutation before. For example cell lines oranimals may be made which carry changes (mutations) in a target RNAsequence, which may be used in assays or as (animal, organoid, etcetera)model systems to study disease, test experimental compounds againstdisease, and the like. The oligonucleotide constructs and methodsaccording to the invention may be used in high throughput screeningsystems (in arrayed format) for making cell banks with a large varietyof target RNAs, for example coding for a large variety of proteinisoforms, for further experimentation, including compound screening,protein engineering and the like. The target RNA may be any cellular orviral RNA sequence, but is more usually a pre-mRNA or an mRNA with aprotein coding function.

Purely for ease of reference, and without the intention to limit theinvention, the following Table 1 is provided to illustrate the potentialcodon changes that can be brought about by adenosine deaminase editingdirected by oligonucleotides of the invention. Table 1 particularlyshould not be interpreted as a limitation of the applicability of theinvention to coding sequences in any RNA; as pointed out already, theinvention can be practised on any RNA target comprising an adenosine,whether in a coding region, an intron, a non-coding exon (such as a 5′-or 3′ untranslated region), in miRNAs, tRNAs, rRNAs and so on. To avoidany misunderstanding about the width of the applicability, changes thatare inconsequential (‘silent’) from a coding perspective may still altergene expression of a certain protein as some codons for the same aminoacid may be more preferred than others and may lead, for instance, todifferent transcription stability or translation efficiency, causing theencoded protein to become more or less abundant than without the change.

TABLE 1 Target codon Amino acid Corrected codon Amino acid AAA Lys GAAGlu AGA Arg AAG Lys GGA Gly AGG Arg GAG Glu GGG Gly AAC Asn GAC Asp AGCSer GGC Gly AAG Lys GAG Glu AGG Arg GGG Gly AAU Arg GAU Asp AGU Ser GGUGly ACA Thr GCA Ala ACG Thr GCG Ala ACC Thr GCC Ala ACG Thr GCG Ala ACUThr GCU Ala AGA Arg GGA Gly AGG Arg GGG Gly AGC Ser GGC Gly AGG Arg GGGGly AGU Ser GGU Gly AUA Ile GAU Asp AUG Met GUG Val AUC Ile GUC Val AUGMet GUG Val AUU Ile GUU Val CAA Gln CGA Arg CAG Gln CGG Arg CAC His CGCArg CAG Gln CGG Arg CAU His CGU Arg CCA Pro CCG Pro CGA Arg CGG Arg CUALeu CUG Leu GAA Glu GGA Gly GAG Glu GGG Gly GCA Ala GCG Ala GUA Val GUGVal GGA Gly GGG Gly GAC Asp GGC Gly GAG Glu GGG Gly GAU Asp GGU Gly UAAStop UGA Stop UAG Stop UGG Trp UCA Ser UCG Ser UGA Stop UGG Trp UUA LeuUUG Leu UAC Tyr UGC Cys UAG Stop UGG Trp UAU Tyr UGU Cys

Particularly interesting target adenosines for editing usingoligonucleotides according to the invention are those that are part ofcodons for amino acid residues that define key functions, orcharacteristics, such as catalytic sites, binding sites for otherproteins, binding by substrates, localization domains, for co- orpost-translational modification, such as glycosylation, hydroxylation,myristoylation, protein cleavage by proteases (to mature the proteinand/or as part of the intracellular routing), and so forth.

A host of genetic diseases are caused by G-to-A mutations, and these arepreferred target diseases because adenosine deamination at the mutatedtarget adenosine will reverse the mutation to wild-type. However,reversal to wild-type may not always be necessary to obtain a beneficialeffect. Modification of an A to a G in a target may also be beneficialif the wild-type nucleotide is other than a G. In certain circumstancesthis may be predicted to be the case, in others this may require sometesting. In certain circumstances, the modification from an A in atarget RNA to a G where the wild-type is not a G may be silent (nottranslated into a different amino acid), or otherwise non-consequential(for example an amino acid is substituted but it constitutes aconservative substitution that does not disrupt protein structure andfunction), or the amino acid is part of a functional domain that has acertain robustness for change. If the A-to-G transition brought about byediting in accordance with the invention is in a non-coding RNA, or anon-coding part of an RNA, the consequence may also be inconsequentialor less severe than the original mutation. Those of ordinary skill inthe art will understand that the applicability of the current inventionis very wide and is not even limited to preventing or treating disease.The invention may also be used to modify transcripts to study the effectthereof, even if, or particularly when, such modification induces adiseased state, for example in a cell or a non-human animal model.

Preferred examples of genetic diseases that can be prevented and/ortreated with oligonucleotides according to the invention are any diseasewhere the modification of one or more adenosines in a target RNA willbring about a (potentially) beneficial change.

Transcribed RNA sequences that are potential target RNA sequencesaccording to the invention, containing mutations of particular interestinclude, but are not limited to those transcribed from the CFTR gene(the cystic fibrosis transmembrane conductance regulator), dystrophin,huntingtin, neurofibromin 1, neurofibromin 2, the δ-globin chain ofhaemoglobin, CEP290 (centrosomal protein 290 kDa), the HEXA gene of theδ-hexosaminidase A, and any one of the Usher genes (e.g. USH2B encodingUsherin) responsible for a form of genetic blindness called Ushersyndrome. A more extensive list is presented further below. The targetsequence will be selected accordingly, and the oligonucleotide constructwill include the desired modification in order to correct the mutation.

Those skilled in the art of CF mutations recognise that between 1000 and2000 mutations are known in the CFTR gene, including R117H, G542X,G551D, R553X, W1282X, and N1303K.

In general, mutations in any target RNA that can be reversed usingoligonucleotide constructs according to the invention are G-to-Amutations, in the case of adenosine deaminase recruitment, and U-to-Cmutations in the case of cytidine deaminase recruitment, andoligonucleotide constructs can be designed accordingly. Mutations thatmay be targeted using oligonucleotide constructs according to theinvention also include C to A, U to A (T to A on the DNA level) in thecase of recruiting adenosine deaminases, and A to C and G to C mutationsin the case of recruiting cytidine deaminases. Although RNA editing inthe latter circumstances may not necessarily revert the mutation towild-type, the edited nucleotide may give rise to an improvement overthe original mutation. For example, a mutation that causes an in framestop codon—giving rise to a truncated protein, upon translation—may bechanged into a codon coding for an amino acid that may not be theoriginal amino acid in that position, but that gives rise to a (fulllength) protein with at least some functionality, at least morefunctionality than the truncated protein.

The target sequence is endogenous to the eukaryotic, preferablymammalian, more preferably human cell. Thus the target sequence is not,for instance, a transgene or a marker gene which has been artificiallyintroduced at some point in the cell's history, but rather is a genethat is naturally present in the cell (whether in mutant or non-mutantform).

The invention is not limited to correcting mutations, as it may insteadbe useful to change a wild-type sequence into a mutated sequence byapplying oligonucleotides according to the invention. One example whereit may be advantageous to modify a wild-type adenosine is to bring aboutskipping of an exon, for example by modifying an adenosine that happensto be a branch site required for splicing of said exon. Another exampleis where the adenosine defines or is part of a recognition sequence forprotein binding, or is involved in secondary structure defining thestability of the RNA. As noted above, therefore, the invention can beused to provide research tools for diseases, to introduce new mutationswhich are less deleterious than an existing mutation, etc.

The amount of oligonucleotide to be administered, the dosage and thedosing regimen can vary from cell type to cell type, the disease to betreated, the target population, the mode of administration (e.g.systemic versus local), the severity of disease and the acceptable levelof side activity, but these can and should be assessed by trial anderror during in vitro research, in pre-clinical and clinical trials. Thetrials are particularly straightforward when the modified sequence leadsto an easily-detected phenotypic change. It is possible that higherdoses of oligonucleotide could compete for binding to a nucleic acidediting entity (e.g. ADAR) within a cell, thereby depleting the amountof the entity which is free to take part in RNA editing, but routinedosing trials will reveal any such effects for a given oligonucleotideand a given target.

One suitable trial technique involves delivering the oligonucleotideconstruct to cell lines, or a test organism and then taking biopsysamples at various time points thereafter. The sequence of the targetRNA can be assessed in the biopsy sample and the proportion of cellshaving the modification can easily be followed. After this trial hasbeen performed once then the knowledge can be retained and futuredelivery can be performed without needing to take biopsy samples.

A method of the invention can thus include a step of identifying thepresence of the desired change in the cell's target RNA sequence,thereby verifying that the target RNA sequence has been modified. Thisstep will typically involve sequencing of the relevant part of thetarget RNA, or a cDNA copy thereof (or a cDNA copy of a splicing productthereof, in case the target RNA is a pre-mRNA), as discussed above, andthe sequence change can thus be easily verified. Alternatively thechange may be assessed on the level of the protein (length,glycosylation, function or the like), or by some functional read-out,such as a(n) (inducible) current, when the protein encoded by the targetRNA sequence is an ion channel, for example. In the case of CFTRfunction, an Ussing chamber assay or an NPD test in a mammal, includinghumans, are well known to a person skilled in the art to assessrestoration or gain of function.

After RNA editing has occurred in a cell, the modified RNA can becomediluted over time, for example due to cell division, limited half-lifeof the edited RNAs, etc. Thus, in practical therapeutic terms a methodof the invention may involve repeated delivery of an oligonucleotideconstruct until enough target RNAs have been modified to provide atangible benefit to the patient and/or to maintain the benefits overtime.

Oligonucleotides of the invention are particularly suitable fortherapeutic use, and so the invention provides a pharmaceuticalcomposition comprising an oligonucleotide of the invention and apharmaceutically acceptable carrier. In some embodiments of theinvention the pharmaceutically acceptable carrier can simply be a salinesolution. This can usefully be isotonic or hypotonic, particularly forpulmonary delivery. The invention also provides a delivery device (e.g.syringe, inhaler, nebuliser) which includes a pharmaceutical compositionof the invention.

The invention also provides an oligonucleotide of the invention for usein a method for making a change in a target RNA sequence in a mammalian,preferably human cell, as described herein. Similarly, the inventionprovides the use of an oligonucleotide construct of the invention in themanufacture of a medicament for making a change in a target RNA sequencein a mammalian, preferably human cell, as described herein.

The invention also relates to a method for the deamination of at leastone specific target adenosine present in a target RNA sequence in acell, said method comprising the steps of: providing said cell with anAON according to the invention; allowing uptake by the cell of said AON;allowing annealing of said AON to the target RNA sequence; allowing amammalian ADAR enzyme comprising a natural dsRNA binding domain as foundin the wild type enzyme to deaminate said target adenosine in saidtarget RNA sequence to an inosine; and optionally identifying thepresence of said inosine in the RNA sequence. Introduction of the AONaccording to the present invention into the cell is performed by generalmethods known to the person skilled in the art. After deamination theread-out of the effect (alteration of the target RNA sequence) can bemonitored through different ways. Hence, the identification step ofwhether the desired deamination of the target adenosine has indeed takenplace depends generally on the position of the target adenosine in thetarget RNA sequence, and the effect that is incurred by the presence ofthe adenosine (point mutation, early stop codon, aberrant splice site,alternative splice site, misfolding of the resulting protein, etc.).Hence, in a preferred aspect, depending on the ultimate deaminationeffect of A-to-I conversion, the identification step comprises:sequencing the target RNA; assessing the presence of a functional,elongated, full length and/or wild type protein when said targetadenosine is located in a UGA or UAG stop codon, which is edited to aUGG codon through said deamination; assessing the presence of afunctional, elongated, full length and/or wild-type protein when twotarget adenosines are located in a UAA stop codon, which is edited to aUGG codon through the deamination of both target adenosines; assessingwhether splicing of the pre-mRNA was altered by said deamination; orusing a functional read-out, wherein the target RNA after saiddeamination encodes a functional, full length, elongated and/or wildtype protein. In the event that there is a UAA stop codon it means thatboth adenosines need to be deaminated. Hence, the invention also relatesto oligonucleotides and methods wherein two adenosines that are next toeach other are co-deaminated by an RNA editing enzyme such as ADAR. Inthis particular case, the UAA stop codon is converted into a UGGTrp-encoding codon (see Table 1). Because the deamination of theadenosine to an inosine may result in a protein that is no longersuffering from the mutated A at the target position, the identificationof the deamination into inosine may also be a functional read-out, forinstance an assessment on whether a functional protein is present, oreven the assessment that a disease that is caused by the presence of theadenosine is (partly) reversed. The functional assessment for each ofthe diseases mentioned herein will generally be according to methodsknown to the skilled person. When the presence of a target adenosinecauses aberrant splicing, the read-out may be the assessment of whetherthe aberrant splicing is still taking place, or not, or less. On theother hand, when the deamination of a target adenosine is wanted tointroduce a splice site, then similar approaches can be used to checkwhether the required type of splicing is indeed taking place. A verysuitable manner to identify the presence of an inosine after deaminationof the target adenosine is of course RT-PCR and sequencing, usingmethods that are well-known to the person skilled in the art.

The present invention relates to an antisense oligonucleotide (AON)capable of forming a double stranded complex with a target RNA sequencein a cell, preferably a human cell, for the deamination of a targetadenosine in the target RNA sequence by an ADAR enzyme present in thecell, said AON comprising a Central Triplet of 3 sequential nucleotides,wherein the nucleotide directly opposite the target adenosine is themiddle nucleotide of the Central Triplet, wherein 1, 2 or 3 nucleotidesin said Central Triplet comprise a sugar modification and/or a basemodification; with the proviso that the middle nucleotide does not havea 2′-O-methyl modification. The sugar and/or base modification of thenucleotides of the present invention render the AON more stable and/orcause an improved induction of deamination of the target adenosine ascompared to AONs not carrying the sugar and/or base modification. In apreferred embodiment, the non-complementary nucleotide that is directlyopposite the target adenosine when the double stranded complex isformed, is a cytidine. This cytidine, together with the nucleotides thatare directly 5′ and 3′ of it in the AON together form the CentralTriplet as defined herein. Although there may be additional mismatchesbetween the AON and the target RNA sequence outside the Central Triplet,the cytidine in the centre of the Central Triplet forms at least onemismatch with the target adenosine in the target sequence such that itcan be edited by the ADAR present in the cell. The cell is preferably ahuman cell and the ADAR is preferably a human ADAR, more preferably anendogenous ADAR in said cell without the need to over-express it byrecombinant means. In any event, the middle nucleotide in the CentralTriplet does not have a 2′-O-methyl modification, allowing the cellularRNA editing enzyme(s) to act. In one embodiment, 1 or 2 nucleotides inthe Central Triplet other than the middle nucleotide are replaced by aninosine. This may be preferred to allow a better fit with the ADARenzyme. In yet another preferred embodiment, the AON does not comprise aportion that is capable of forming an intramolecular stem-loop structurethat is capable of binding a mammalian ADAR enzyme.

As exemplified in FIGS. 1, 2A-D and 4A-B, the Central Triplet isdepicted as YXZ or as XXY. Different base modifications are provided.The skilled person understands that these base modifications are to acertain level dependent on the nucleotides opposite the nucleotides inthe Central Triplet. Hence, in the example of YXZ, wherein X is themiddle nucleotide opposite the target adenosine, the X may be cytidine,5-methylcytidine, 5-hydroxymethylcytidine, uridine, or pyrrolocytidine,whereas the Y and/or Z may be inosine, 5-methylcytidine,pyrrolocytidine, pseudouridine, 4-thiouridine, thienouridine,2-aminopurine, 2,6-diaminopurine, thienoguanosine, 5-methoxyuridine,dihydrouridine, 5-hydroxymethylcytidine, 5-methyluridine,8-aza-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine,7-methyladenosine, 8-methyladenosine, 3-deazaadenosine,7-deazaadenosine, 8-azidoadenosine, etc., depending on the nucleotidesopposite the Central Triplet; hence depending on the target RNA sequenceand disease related to that. Ergo, in a preferred embodiment, said basemodification is selected from the group consisting of 2-aminopurine,2,6-diaminopurine, 3-deazaadenosine, 7-deazaadenosine,7-methyladenosine, 8-azidoadenosine, 8-methyladenosine,5-hydroxymethylcytidine, 5-methylcytidine, Pyrrolocytidine,7-aminomethyl-7-deazaguanosine, 7-deazaguanosine, 7-methylguanosine,8-aza-7-deazaguanosine, thienoguanosine, inosine, 4-thio-uridine,5-methoxyuridine, 5-methyluridine, dihydrouridine, pseudouridine, andthienouridine.

In another preferred embodiment, the sugar modification is selected fromthe group consisting of deoxyribose (i.e. DNA), Unlocked Nucleic Acid(UNA) and 2′-fluororibose. In a particularly preferred aspect, thepresent invention relates to an AON comprising a Central Triplet of 3sequential nucleotides, wherein the nucleotide directly opposite thetarget adenosine is the middle nucleotide of the Central Triplet, andwherein 1, and preferably 2, and even more preferably all 3 nucleotidesin said Central Triplet are DNA nucleotides to render the AON morestable and/or more effective in inducing deamination of the targetadenosine. In another preferred aspect, the remainder of the AONconsists of RNA nucleotides that preferably (but not necessarily) are2′-O-methyl modified. Other stabilizing modifications may be usedoutside the Central Triplet. Other ribose modifications that are quitecompatible with targeted editing in accordance with the invention are2′-O-methoxyethyl (2′-O-MOE), LNA, 2′-F and 2′-NH₂. Differentcombinations of sugar modifications (as listed herein) of thenucleotides outside the Central triplet may be applied. In anotherpreferred aspect, the AON according to the invention comprises at leastone internucleoside linkage modification selected from the groupconsisting of phosphorothioate, 3′-methylenephosphonate (i.e.3′-O-methylphosphonate internucleotide linkage), 5′-methylenephosphonate(i.e. 5′-O-methylphosphonate internucleotide linkage),3′-phosphoroamidate (i.e. N-3′-phosphoroamidate internucleotide linkage)and 2′-5′-phosphodiester (i.e. 2′-5′-phosphodiester internucleotidelinkage). Especially preferred are phosphorothioate linkages. Furtherpreferred AONs according to the invention are AONs wherein the 2, 3, 4,5, or 6 terminal nucleotides of the 5′ and 3′ terminus of the AON arelinked with phosphorothioate linkages, preferably wherein the terminal 5nucleotides at the 5′ and 3′ terminus are linked with phosphorothioatelinkages. As disclosed herein, also the nucleotides within the CentralTriplet may be connected through phosphorothioate linkages, although itappears that there is preferably more than one such linkage present inthe Central Triplet to render the AON stable as well as active in RNAediting. In a further preferred aspect, the AON is annealed to aprotecting sense oligonucleotide (SON) for increased stability and, thisis hypothesized, prolonged activity due to the more stable character ofthe double stranded antisense oligonucleotide+SON complex. The SON doesnot necessarily have to be the same length as the antisenseoligonucleotide. It may be longer, the same length or shorter.

In one preferred aspect, all nucleotides in the AON outside the CentralTriplet comprise a 2′-O-alkyl group such as a 2′-O-methyl group, or a2′-O-methoxyethyl group. Nucleotides outside the Central triplet mayalso be DNA as long as a DNA stretch does not result in a gapmer. Also,the AON is preferably longer than 10, 11, 12, 13, 14, 15, 16 or 17nucleotides, and preferably shorter than 100 nucleotides, morepreferably shorter than 60 nucleotides. In yet another preferredembodiment, the AON comprises 18 to 70 nucleotides, more preferablycomprises 18 to 60 nucleotides, and even more preferably comprises 18 to50 nucleotides. The invention also relates to a pharmaceuticalcomposition comprising the AON according to any one of claims 1 to 12,and a pharmaceutically acceptable carrier.

The oligonucleotide according to the invention is suitably administratedin aqueous solution, e.g. saline, or in suspension, optionallycomprising additives, excipients and other ingredients, compatible withpharmaceutical use, at concentrations ranging from 1 ng/ml to 1 g/ml,preferably from 10 ng/ml to 500 mg/ml, more preferably from 100 ng/ml to100 mg/ml. Dosage may suitably range from between about 1 μg/kg to about100 mg/kg, preferably from about 10 μg/kg to about 10 mg/kg, morepreferably from about 100 μg/kg to about 1 mg/kg. Administration may beby inhalation (e.g. through nebulization), intranasally, orally, byinjection or infusion, intravenously, subcutaneously, intra-dermally,intra-cranially, intramuscularly, intra-tracheally, intra-peritoneally,intra-rectally, by direct injection into a tumor, and the like.Administration may be in solid form, in the form of a powder, a pill, orin any other form compatible with pharmaceutical use in humans.

The invention is particularly suitable for treating genetic diseases,such as cystic fibrosis, albinism, alpha-1-antitrypsin (A1AT)deficiency, Alzheimer disease, Amyotrophic lateral sclerosis, Asthma,ß-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, ChronicObstructive Pulmonary Disease (COPD), Distal Spinal Muscular Atrophy(DSMA), Duchenne/Becker muscular dystrophy, Dystrophic Epidermolysisbullosa, Epidermolysis bullosa, Fabry disease, Factor V Leidenassociated disorders, Familial Adenomatous Polyposis, Galactosemia,Gaucher's Disease, Glucose-6-phosphate dehydrogenase deficiency,Haemophilia, Hereditary Hemachromatosis, Hunter Syndrome, Huntington'sdisease, Hurler Syndrome, Inflammatory Bowel Disease (IBD), Inheritedpolyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhansyndrome, Lynch syndrome, Marfan syndrome, Mucopolysaccharidosis,Muscular Dystrophy, Myotonic dystrophy types I and II,neurofibromatosis, Niemann-Pick disease type A, B and C, NY-eso1 relatedcancer, Parkinson's disease, Peutz-Jeghers Syndrome, Phenylketonuria,Pompe's disease, Primary Ciliary Disease, Prothrombin mutation relateddisorders, such as the Prothrombin G20210A mutation, PulmonaryHypertension, Retinitis Pigmentosa, Sandhoff Disease, Severe CombinedImmune Deficiency Syndrome (SCID), Sickle Cell Anemia, Spinal MuscularAtrophy, Stargardt's Disease, Tay-Sachs Disease, Usher syndrome,X-linked immunodeficiency, Sturge-Weber Syndrome, various forms ofcancer (e.g. BRCA1 and 2 linked breast cancer and ovarian cancer), andthe like.

In some embodiments the oligonucleotide construct can be deliveredsystemically, but it is more typical to deliver an oligonucleotide tocells in which the target sequence's phenotype is seen. For instance,mutations in CFTR cause cystic fibrosis which is primarily seen in lungepithelial tissue, so with a CFTR target sequence it is preferred todeliver the oligonucleotide construct specifically and directly to thelungs. This can be conveniently achieved by inhalation e.g. of a powderor aerosol, typically via the use of a nebuliser. Especially preferredare nebulizers that use a so-called vibrating mesh, including the PARIeFlow (Rapid) or the i-neb from Respironics. The inventors have foundthat inhaled use of oligonucleotide constructs can lead to systemicdistribution of the oligonucleotide construct and uptake by cells in thegut, liver, pancreas, kidney and salivary gland tissues, among others.It is therefore to be expected that inhaled delivery of oligonucleotideconstructs according to the invention can also target these cellsefficiently, which in the case of CFTR gene targeting could lead toamelioration of gastrointestinal symptoms also associated with cysticfibrosis. For other target sequences, depending on the disease and/orthe target organ, administration may be topical (e.g. on the skin),intradermal, subcutaneous, intramuscular, intravenous, oral, ocularinjection, etc.

In some diseases the mucus layer shows an increased thickness, leadingto a decreased absorption of medicines via the lung. One such a diseaseis chronical bronchitis, another example is cystic fibrosis. Variousforms of mucus normalizers are available, such as DNAses, hypertonicsaline or mannitol, which is commercially available under the name ofBronchitol. When mucus normalizers are used in combination with RNAediting oligonucleotide constructs, such as the oligonucleotideconstructs according to the invention, they might increase theeffectiveness of those medicines. Accordingly, administration of anoligonucleotide construct according to the invention to a subject,preferably a human subject is preferably combined with mucusnormalizers, preferably those mucus normalizers described herein. Inaddition, administration of the oligonucleotide constructs according tothe invention can be combined with administration of small molecule fortreatment of CF, such as potentiator compounds for example Kalydeco(ivacaftor; VX-770), or corrector compounds, for example VX-809(lumacaftor) and/or VX-661. Other combination therapies in CF maycomprise the use of an oligonucleotide construct according to theinvention in combination with an inducer of adenosine deaminase, usingIFN-gamma or TNF-alpha.

Alternatively, or in combination with the mucus normalizers, delivery inmucus penetrating particles or nanoparticles can be applied forefficient delivery of RNA editing molecules to epithelial cells of forexample lung and intestine. Accordingly, administration of anoligonucleotide construct according to the invention to a subject,preferably a human subject, preferably uses delivery in mucuspenetrating particles or nanoparticles.

Chronic and acute lung infections are often present in patients withdiseases such as cystic fibrosis. Antibiotic treatments reduce bacterialinfections and the symptoms of those such as mucus thickening and/orbiofilm formation. The use of antibiotics in combination witholigonucleotide constructs according to the invention could increaseeffectiveness of the RNA editing due to easier access of the targetcells for the oligonucleotide construct. Accordingly, administration ofan oligonucleotide construct according to the invention to a subject,preferably a human subject, is preferably combined with antibiotictreatment to reduce bacterial infections and the symptoms of those suchas mucus thickening and/or biofilm formation. The antibiotics can beadministered systemically or locally or both.

For application in for example cystic fibrosis patients theoligonucleotide constructs according to the invention, or packaged orcomplexed oligonucleotide constructs according to the invention may becombined with any mucus normalizer such as a DNase, mannitol, hypertonicsaline and/or antibiotics and/or a small molecule for treatment of CF,such as potentiator compounds for example ivacaftor, or correctorcompounds, for example lumacaftor and/or VX-661.

To increase access to the target cells, Broncheo-Alveolar Lavage (BAL)could be applied to clean the lungs before administration of theoligonucleotide according to the invention.

The inventors of the present invention have shown herein that certainmodifications to AONs targeting RNA and recruiting ADAR enzymes yield inincreased stability and through this, better applicability in in vivosettings. However, stability is one thing. In addition to stabilizingthe AONs by preventing nuclease-mediated degradation, the modifiednucleotides are ideally selected so that they support and preferablyimprove binding and/or catalytic activity of the ADAR enzymes. The knownstructural features of the interaction between the ADAR2 deaminasedomain and a dsRNA substrate (provided in great detail in Matthews etal. 2016. Nat Struct Mol Biol 23:426-433) can be used to rationallyselect modified AONs that best fit into the catalytic center of theADAR2 enzyme. From the (colored) structure disclosed in Matthews et al.(2016) it can be seen that ADAR2 contacts the editing site from theminor groove of the dsRNA helix; FIG. 2 therein, Panel A: View of thestructure of the complex perpendicular to the dsRNA helical axis. Thepink RNA strand is the edited strand, and the blue strand is thecomplementary strand. The flipped-out target base (N) is shown in red.The protein makes contacts with the helix through its minor groove.Panel B: View of structure along the dsRNA helical axis. Panel C:Summary of the contacts between ADAR2 E488Q deaminase domain and RNAduplex. Red color indicates the edited target strand, and blue color thecomplementary strand (equivalent to the AONs presented here). Dashedlines indicate interactions of the amino acid side chains (amino acididentity and position indicated) of the deaminase domain with the RNAstrands. FIG. 5A of Matthews et al. (2016) shows in detail aspace-filling model of the interaction of the nucleotide 5′ to theedited site (on the target strand) and the complementary nucleotide onthe other strand. In the upper panel, the 5′ nucleotide is a uridine,and the interaction to the opposing adenosine is stabilized by theglycine 489 of the ADAR2 deaminase domain. The lower panel shows themodelled interaction of a C (5′ to the edited nucleotide) with acomplementary G. In this case, interaction with the glycine results in aclash with the 2-amino group, which reduces editing efficiency.Replacement of the guanosine base on the opposing strand (or AON) withan inosine would resolve this steric clash.

For base modifications, the inventors of the present invention deducedthat chemical modifications that are projected into the major groove ofthe helix (Sharma and Watts 2015, Future Med. Chem. 7(16), 2221-2242)would interfere with the function of ADAR2 the least. See FIGS. 2A-2Dand examples below for details of the different AONs. For pyrimidinenucleotides, such modifications include substituents at position 4 or 5of the base, including 4-thiouridine (see e.g. ADAR60-13),5-methyluridine (see e.g. ADAR60-14), 5-methoxyuridine (see e.g.ADAR60-26), 5-methylcytidine (see e.g. ADAR60-9, -11 through 16, -20,26, -27 and -29 through -32 and ADAR68-1 through -6) and5-hydroxymethylcytidine (see e.g. ADAR60-28). For purine nucleotides,substituents in the positions 7 and 8 of the base would similarly bedirected into the major groove, including 7-methylguanosine,7-deazaguanosine, 8-aza-7-deazaguanosine,7-aminomethyl-7-deazaguanosine, 7-methyladenosine, 8-methyladenosine,3-deazaadenosine, 7-deazaadenosine, or 8-azidoadenosine (see ADAR60-29,-30, -31, and -32, and ADAR68-1, -2, -3, -4 and -5, respectively).

For modifications of the backbone (i.e. modifications of the ribosylmoiety of the nucleotides or the internucleotide linkages) of the AON,the inventors of the present invention deduced that modifications thatdo not change the A form structure of the RNA helix would only modestlyinterfere with the function of ADAR2. The structural information of theADAR2 deaminase domain also suggests that in certain positions it isimportant to retain chemical groups that are contacted by amino acidside chains of the enzyme, such as phosphate groups and 2′-hydroxylgroups. Modifications that permit the retention of these featuresinclude UNA (see e.g. ADAR60-8 and -9), 3′-methylenephosphonate,5′-methylenephosphonate, 3′-phosphoroamidate, or 2′-5′ phosphodiestermodifications (see ADAR60-22, -23, -24 and -25, respectively). In somecases, a direct interaction by ADAR2 is not present for a givensubstituent on a specific nucleotide. In such cases, other chemicalgroups can be used that protect the AON from nucleases, but are smallenough not to cause steric interference with ADAR2, e.g. a 2′-fluoromodification or 2′-H (deoxy) as it is not larger than the naturallyoccurring hydroxyl group (see e.g. ADAR60-7).

Modified nucleotides can be selected not only to provide enhancedprotection from nucleases and minimal interference to ADAR2 binding, butalso to specifically increase functionality of ADAR2. Specificnucleotide modifications may in particular be necessary to enhance theediting activity on substrate RNAs where the target sequence is notoptimal for ADAR editing. Previous work has established that certainsequence contexts are more amenable to editing. For example, the targetsequence 5′-UAG-3′ (with the target A in the middle) contains the mostpreferred nearest-neighbor nucleotides for ADAR2, whereas a 5′-CAA-3′target sequence is disfavored (Schneider et al. 2014). The structuralanalysis of ADAR2 deaminase domain hints at the possibility of enhancingediting by careful selection of the nucleotides that are opposite to thetarget trinucleotide (Matthews et al. 2016). For example, the 5′-CAA-3′target sequence, paired to a 3′-GCU-5′ sequence on the opposing strand(with the A-C mismatch formed in the middle), is disfavored because theamino group of the guanosine base sterically clashes with an amino acidside chain of ADAR2. However, it is postulated that a smaller nucleobaselacking the amino group, such as inosine, could fit better into thisposition without causing steric clashes, while still retaining thebase-pairing potential to the opposing cytosine (see e.g. ADAR60-10through -19, and -26, -27, -28 and -32). An inosine could also be usedwithin a different sequence context. For example, a target sequence5′-UAG-3′ could be paired with the AON Central Triplet sequence5′-CCI-3′, thus forming a U-I wobble base pair that can provideincreased flexibility that is needed for the target adenosine to flipout of the helix into the active site of the enzyme (see e.g. ADAR68-6).

Backbone modifications can also have additional benefits beyond nucleaseprotection, such as providing increased flexibility for the nucleotidessurrounding the edited site, which has been suggested as an importantfactor for correctly positioning the substrate in the active site of theenzyme. UNA is one such backbone modification, as the open structure ofthe sugar moiety allows increased movement (see ADAR60-8 and -9).Conversely, modifications that could enhance activity of suboptimalsequences also include the use of backbone modifications that force itinto a conformation that favors editing.

EXAMPLES Example 1. Chemical Modifications in AONs Targeting HumanSERPINA1 RNA to Increase Stability

Unpublished patent application PCT/EP2017/065467 describes a method fortargeted A-to-I editing by the use of antisense oligonucleotides (AONs).Whereas most of the nucleotides in these AONs are 2′-O-methylated RNAand have phosphorothioate linkages, they contain a few, usually 3,unmodified RNA nucleotides (the ‘Central Triplet’) in the portion of theAON with the middle nucleotide directly opposed to the target adenosineand its 2 neighbouring nucleotides. This is because it had previouslybeen shown that A-to-I editing is strongly inhibited when thenucleotides of the Central Triplet are 2′-O-methylated (Vogel et al.2014). However, AONs containing unmodified RNA nucleotides areinherently biologically unstable due to nucleases that can hydrolyze theresidues in these positions. This may be a disadvantage for efficientuse of the AONs as e.g. therapeutic agents, as they may be degradedprior to reaching their targets.

The inventors of the present invention investigated the stability of theAONs with unmodified RNA nucleotides at the Central Triplet, and whetherthe stability of such AONs could be increased by using chemicalmodifications other than 2′-O-methylation.

First, as a non-limiting example, the target that was selected for RNAediting was the c.1096G>A mutation in the SERPINA1 RNA, which is thecause of A1AT-deficiency (A1ATD). A1ATD is an example of a diseasetarget for RNA editing using the approach as outlined herein. In FIG. 1Aand FIG. 1B, the complementarity of exemplary AONs to the SERPINA1target is illustrated schematically. The ADAR60-1 complementarity to thetarget sequence is shown in FIG. 1A, whereas the Central Triplet in theADAR60 series (5′-YXZ-3′) is indicated as such in FIG. 1B. A number ofAONs targeting the mutant RNA with varying modifications in the backboneas well as in the bases of the nucleotides of the Central Triplet weretested for stability by incubating them in cell culture medium (MEM)containing 15% Fetal Bovine Serum (FBS) at +37° C. for 30 min (RX). Asnegative controls, AONs were incubated in Phosphate Buffered Saline(PBS; CTL). The samples were then resolved in denaturing polyacrylamidegels (15% PAGE gel with 8 M urea), which were subsequently stained withtoluidine blue to visualize the AONs and fragments thereof. The AONswith their respective sequences and their modifications are indicated inFIGS. 2A-2D. FIGS. 3A-3C show the results of the stability assays usingthe AONs of the ADAR60 series. ADAR60-1, containing an unmodified5′-UCG-3′ RNA Central Triplet, is efficiently cleaved (˜degraded) within30 min in the presence of FBS (to approximately 50%). Some degradationis even apparent when the AON is incubated in control buffer. The rateof degradation is not reduced when the location of the three RNAnucleotides is shifted (ADAR60-3). Importantly, lowering the number ofunmodified RNA nucleotides to 2 (instead of 3) results in a reduction indegradation (ADAR60-2). A fully 2′-O-methylated AON, which here servesas a negative control, remains stable under these conditions (ADAR60-4),as does an AON where the Central Triplet has been changed to DNAnucleotides (ADAR60-6). This shows at least that when the CentralTriplet is not 2′-O-methylated, the AON is prone to degradation and thatsuch can be resolved by adding at least one 2′-O-methylated nucleotideto the Central Triplet, or amending the entire Central Triplet to DNAnucleotides. Another modification of the sugar, replacement of RNA byunlocked nucleic acid (UNA) in 2 of the 3 positions of the CentralTriplet also results in increased stability (ADAR60-8), and even more sowhen combined with the base modification 5-methylcytosine in the middleof the triplet (ADAR60-9). Inclusion of one phosphorothioate linkageafter the first position of the Central Triplet (5′-U*CG-3′ in which theasterisk represents the phosphorothioate linkage) does not increasestability appreciably (ADAR60-5). See example 2 below for furtherexperiments towards increasing numbers of phosphorothioate linkages.

Replacement of guanosine with inosine also results in noticeablereduction in degradation (ADAR60-10). When this modification is combinedwith the replacement of cytidine for 5-methylcytidine (ADAR60-11),stability remains and is further increased when both modifications arecombined with the replacement of uridine for pseudouridine (ADAR60-12).

Replacing all 3 nucleotides of the Central Triplet with modifiednucleotides appeared to yield the greatest effect on stability. Theexact modifications in each position can be varied, and this greatlyaffects the stability of the AON. In AONs otherwise similar toADAR60-12, replacement of the pseudouridine with 4-thiouridine(ADAR60-13) or 2,6-diaminopurine (ADAR60-16) results in AONs ofcomparable stability, while 5-methyluridine (ADAR60-14) in contrastreduces stability, and thienouridine (ADAR60-15) causes almost completedegradation of the AON, even more so than with the unmodified RNAtriplet.

Replacement of the middle C in the Central Triplet with pyrrolocytidineresults in an even more significant stabilization than with5-methylcytidine: AONs with pyrrolocytidine and inosine are nearly fullystable under these conditions regardless of whether they contain apseudouridine or unmodified uridine in the first position of the triplet(ADAR60-18 and ADAR60-17, respectively). Replacement of the uridine withthienouridine (ADAR60-19) again results in reduced stability also inthis context, but not to the same extent as with ADAR60-15.

Similar to the other two positions of the Central Triplet, the lastposition can also be modified with different base modifications toachieve stabilization. Here, instead of inosine, thienoguanosine wasused in combination with pseudouridine and either 5-methylcytidine orpyrrolocytidine (ADAR60-20 and ADAR60-21, respectively). Both AONs arefully stable under these conditions; the increased stability withthienoguanosine is noticeable when comparing with the otherwise similarADAR60-12 and ADAR60-20.

It is envisioned that additional combinations to achieve a similareffect for increased stabilization are replacement of e.g. guanosine by7-methylguanosine, 7-deazaguanosine, 8-aza-7-deazaguanosine, or7-aminomethyl-7-deazaguanosine (see ADAR60-29, -30, -31 and -32,respectively), or that of cytidine by 5-hydroxymethylcytosine (seeADAR60-28), or that of uridine with 5-methoxyuridine or dihydrouridine(see ADAR60-26 and -27, respectively). Stabilization was also achievedby sugar and backbone modifications of the Central Triplet, including2′-fluoro, 3′-methylenephosphonate, 5′-methylenephosphonate,3′-phosphoroamidate, or 2′-5′ phosphodiester modifications (2′ to 5′backbone linkages; see ADAR60-7, -22, -23, -24 and -25, respectively).

The results shown in this example indicate that not every modificationis useful for reducing degradation of RNA-editing antisenseoligonucleotides but many modifications do add to the stability, andthat the modifications in and possibly around the Central Triplet shouldbe carefully selected. Modifying the nucleotides in the Central Tripletwith specified chemistries surely increases the stability of theoligonucleotide. All in all, the results presented in this example showthat (combinations of) modifications of the nucleotides in the CentralTriplet can improve stability, and that modification of several bases atonce results in incremental stabilization.

Example 2. Phosphorothioate Linkages in AONs Targeting Mouse Ldua RNA toIncrease Stability

In the previous example it was shown that inclusion of onephosphorothioate linkage after the first position within the CentralTriplet did not increase stability appreciably (FIG. 3A; ADAR60-5). Toinvestigate this in more depth, it was tested whether inclusion of morephosphorothioate linkages within the Central Triplet region couldnevertheless have an influence and increase stability.

For this, the inventors of the present invention selected Hurlersyndrome as the model system, also known as mucopolysaccharidosis typeI-Hurler (MPS I-H), which in humans is caused by the c.1205G>A (W402X)mutation of the IDUA gene. The target sequence in the mutated human genewould be 5′-UAG-3′ (with the target A in the middle, see FIG. 4A), andthe Central Triplet of the AON would then be 5′-CCA-3′ (including thecentral, mismatched C; see FIG. 4B for the 5′-XXY-3′ notation) or5′-CUA-3′. A mouse model for this mutation also exists (W392X), whereinthe ldua gene is mutated. The Hurler mouse model was also used foradditional in vivo experiments (see below). Oligonucleotides having theserial number ADAR65 (see FIGS. 2A-2D) were designed to target themutated mouse ldua gene. ADAR65-1, ADAR65-18, ADAR65-20, ADAR65-21 andADAR65-22 were tested to see whether additional phosphorothioatelinkages would add to the stability of the oligonucleotide.Surprisingly, inclusion of 3 or more phosphorothioates in the CentralTriplet region did give a stability increase of the AON (FIG. 5 ).ADAR65-1 has no additional phosphorothioate linkages in the CentralTriplet region and is clearly degraded, whereas ADAR65-22 has 6phosphorothioate linkages in and around the Central Triplet region andis clearly more stable, even after incubation of 18 h in FBS. Similarlythe stability of ADAR65-20 and ADAR65-21 that respectively have 5 and 3phosphorothioate linkages in the Central Triplet region, was increasedwhen compared to ADAR65-18 that (like ADAR65-1) has no additionalphosphorothioates in the Central Triplet region, except for the terminallinkages of the oligonucleotide.

Base modifications such as 5-methylcytidine and deoxy 2-aminopurinetogether with ribose modification such deoxyribose (DNA), 2′-O-methyl,2′-fluoro and a phosphate backbone (phosphorothioate linkages) werecombined. ADAR65-13, -14, -15, -16, -19, -20, -21, -22, -23, -24, -25,-26, -27, -28, -29, and -30 (FIGS. 2A-2D) were tested for stabilityusing the same assay as described above. The results that are depictedin FIG. 6 show that a variety of combinations can be used to obtainstability, even after 18 h incubation in FBS.

Further to this, AONs with either RNA (ADAR93-2) or DNA (ADAR93-6,ADAR93-8, ADAR93-9) in the Central Triplet and varying numbers ofphosphorothioate nucleotides (28 in ADAR93-2, 8 in ADAR93-6, 21 inADAR93-8, 22 in ADAR93-9) were analyzed for stability in variousbiological solutions. PBS was used as a negative control. To mimic thephysiological conditions that the AONs may be subjected to in blood andthe central nervous system, the AONs were, respectively, incubated incell culture medium (DMEM) containing 15% FBS or Single Donor HumanCerebrospinal Fluid (CSF). In order to assess whether the acidicmicroenvironment of a lysosome and nucleases therein would affect AONstability, oligonucleotides were also incubated in Mixed Gender HumanLiver Lysosomes (Lyso). 200 pmol of AON was incubated in each conditionfor 2 h, 24 h or 3 days, after which the samples were resolved indenaturing polyacrylamide gels (12% PAGE gel with 8 M urea). The gelswere stained with toluidine blue solution and destained in water tovisualize the AONs and fragments thereof. The results are shown in FIG.7 and indicate (again) that oligonucleotides that contain RNA withoutmodifications in the Central Triplet (ADAR93-2) are very prone todegradation in FBS, CSF and Lyso because almost all of theoligonucleotides are rapidly digested under these differentcircumstances, whereas they remain relatively stable in PBS alone.However, changing the Central Triplet to include two or three DNAnucleotides significantly increases the stability of the oligonucleotidein the three different environments, even up to three days.

Antisense oligonucleotides ADAR68, ADAR68-1, -2, -3, -4, and -5 (seeFIGS. 2A-2D) potentially targeting the c.1205G>A (W402X) mutation of thehuman IDUA gene were also designed. The modified Central Triplet can becomposed of e.g. two 5-methylcytidines in combination with a nucleotidereplacing the adenosine, such as 7-methyladenosine, 8-methyladenosine,3-deazaadenosine, 7-deazaadenosine, or 8-azidoadenosine (ADAR68-1, -2,-3, -4 and -5 in FIGS. 2A-2D). However, the adenosine can also bereplaced by a modified base that can form a wobble base pair with theopposing uridine, such as inosine (ADAR68-6). These AONs are tested forstability as well as for their ability to edit RNA of a target sequence.

Example 3. RNA Editing Activity of AONs with Combined Chemical BaseModifications in the Central Triplet

Nucleotide phosphodiester, sugar and base modifications are used toincrease the stability of oligonucleotides against nucleases.Importantly, it was envisioned by the inventors of the present inventionthat such chemical nucleotide modifications may also be used for otherpurposes such as increased binding of AONs to their target RNA, to getbetter recognition and/or to increase the enzymatic activity of proteinsacting on double-stranded complexes of AONs with their specific targetRNA sequences.

The inventors first wanted to know whether a combination of nucleotidebase chemical modifications in the Central Triplet of an AON couldenhance the editing activity of ADAR2. For this, ADAR60-1 and ADAR60-15(see FIGS. 2A-2D and 3A-3C) were used to investigate such influence onenhancing editing activity of ADAR2 enzyme on a human mutated SERPINA1target RNA molecule. These two AONs share the same sequence and length,and both have the chemical phosphorothioate modifications of phosphategroups as well as 2′-O-methyl modifications of ribose sugars outside theCentral Triplet. Nucleotides in the Central Triplet of ADAR60-1 areunmodified, whereas the nucleotide bases in the Central Triplet ofADAR60-15 are chemically modified as depicted in FIGS. 2A-2D. The5-methylcytidine is opposite the target adenosine for editing. BothADAR60-1 and ADAR60-15 were tested in an in vitro editing assay usingHEK293 cell lysates with overexpressed isoform 2 of ADAR2 (ADAR2a), andSERPINA1 target RNA carrying the c.1096G>A mutation. ADAR60-15 withoutcell lysate, and HEK293 cell lysate with overexpressed ADAR2a butwithout oligonucleotides were used as negative controls. The SERPINA1template ssRNA was obtained by in vitro transcription of 500 ngSERPINA1mut DNA sequence (amplified by PCR from SERPINA1mut gBlocks®gene fragments) using a MEGAscript Kit (Life Technology) applyinggeneral technologies known to the person skilled in the art andfollowing the manufacturer's protocol, and using the T1promoterF1forward primer (5′-GCGAAGCTTAATACGACTC-3′; SEQ ID NO:3) and theSerpina1-R2 reverse primer (5′-CCATGAAGAGGGGAGACTTC-3′; SEQ ID NO:4).The SERPINA1mut DNA template has the following sequence (with the targetadenosine in bold and underlined):

(SEQ ID NO: 5) 5′-GCGAAGCTTAATACGACTCACTATAGGGTCAACTGGGCATCACTAAGGTCTTCAGCAATGGGGCTGACCTCTCCGGGGTCACAGAGGAGGCACCCCTGAAGCTCTCCAAGGCCGTGCATAAGGCTGTGCTGACCATCGAC A AGAAAGGGACTGAAGCTGCTGGGGCCATGTTTTTAGAGGCCATACCCATGTCTATCCCCCCCGAGGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAACAAAATACCAAGTCTCCCCTCTTCATGG-3′

The target ssRNA has the following sequence (with the target adenosinein bold and underlined):

(SEQ ID NO: 6) 5′-UCAACUGGGCAUCACUAAGGUCUUCAGCAAUGGGGCUGACCUCUCCGGGGUCACAGAGGAGGCACCCCUGAAGCUCUCCAAGGCCGUGCAUAAGG CUGUGCUGACCAUCGAC AAGAAAGGGACUGAAGCUGCUGGGGCCAUGUUUUUAGAGGCCAUACCCAUGUCUAUCCCCCCCGAGGUCAAGUUCAACAAACCCUUUGUCUUCUUAAUGAUUGAACAAAAUACCAAGUCUCCCCUCUUCA UGG-3′

The target ssRNA was gel purified using denaturing polyacrylamide gelelectrophoresis. To obtain the lysates, HEK293 cells were firsttransfected overnight with 500 ng ADARB1 expression plasmid (OriGene)using Lipofectamine 3000. Cells were then lysed using Lysis-M reagent.200 nM AONs and 90 nM SERPINA1 template ssRNA were pre-incubatedtogether in an in vitro editing assay buffer for 30 min at 30° C. Afterpre-incubation the cell lysates were added (10 μl) and the reaction mixwas incubated for 30 min at 30° C. and subsequently for 30 min at 37° C.Targeted RNAs were then extracted by phenol chloroform extraction andreverse transcribed using Maxima RT Reagents, using the protocol of themanufacturer, and using the Serpina1-F2 forward primer(5′-TCAACTGGGCATCACTAAGG-3′; SEQ ID NO:7) and the Serpina1-R2 reverseprimer (see above). Then, cDNAs were amplified by PCR and analysed bySanger sequencing using the Serpina1-R1 primer(5′-CATGAAGAGGGGAGACTTGG-3′; SEQ ID NO:8).

FIGS. 8A-8D show that when ADAR60-1 is incubated with the SERPINA1mutssRNA target, no A-to-G change can be detected (FIG. 8A, positionindicated by an arrow). In contrast however, the use of ADAR60-15 (FIG.8B) shows a clearly detectable and significant A-to-G change, confirmingediting of the target SERPINA1 RNA. The negative control reactions(FIGS. 8C and 8D) show no A-to-G editing. It is concluded that thenucleotide base chemical modifications in ADAR60-15 can enhance the RNAediting activity of ADAR2 in comparison to an oligonucleotide that hasno such modifications (ADAR60-1).

Example 4. RNA Editing of a Non-Sense Mutation in GFP Target RNA Usingan AON Comprising a 2-Aminopurine Modification in the Central Triplet

RNA editing was also investigated in cells containing an expressionconstruct encoding a Green Fluorescent Protein (GFP), stably integratedinto the cellular genome (see WO 2016/097212 for details andconstructs). In this construct, a stop codon (TAG) was introduced atcodon position 57, resulting in a triplet UAG in the target RNA. Editingof the RNA at the adenosine in the middle of this triplet wouldeventually result in a Trp encoding codon and then in the expression ofa full length protein. The construct and the cell line (cells containinga stably integrated GFP W57X plasmid) were generated using techniquesknown to the person of ordinary skill in the art. To ensure that noother adenosines in the target RNA are edited, only the threenucleotides in the AON opposite the stop codon (the 5′-CCA-3′ in theoligonucleotide, with the mismatched C in the middle, see ADAR59-2 andADAR59-10, FIGS. 2A-2D) do not contain a 2′-O-methyl modification,whereas all other nucleotides in the antisense oligonucleotide do.Furthermore, the terminal five nucleotides on each side of all testedoligonucleotides are connected by phosphorothioate linkages, whereas theremaining linkages were normal linkages. In ADAR59-10 the A in theCentral Triplet of the oligonucleotide is chemically modified to be a2-aminopurine, whereas ADAR59-2 does not have that particularmodification. FIG. 5B in Matthews et al. (2016) shows that a2-aminopurine modification in this position results in reduced (but notabolished) activation. However, the modification may still enhance RNAediting indirectly, for example by potentially providing enhancednuclease resistance or enhancing cellular processes.

Stable GFP W57X containing cells were transfected first with 1 μg ofADAR2 overexpression plasmid (see the previous example), followed bytransfection of 150 nM of each of the oligo's in separate batches 24 hlater (both with Lipofectamine 2000). Then, 24 h after AON transfection,RNA was isolated for cDNA synthesis and subsequent RT-PCR and sequencingwas performed to reveal whether RNA editing had occurred.

As shown in FIG. 9 , the editing level observed with ADAR59-10 wasimproved over that of the control without the 2-aminopurine modification(ADAR59-2). This shows that, despite a negative effect on catalyticactivity itself, an AON modification can have a positive effect onoverall editing levels by indirect means. By combining such amodification with other modified nucleotides that support enhancedstability and/or editing activity (as disclosed herein), it is nowpossible to produce stable AONs that efficiently activate RNA editing.

Example 5. Editing of a Non-Sense Mutation in GFP Target RNA Using anAON Comprising DNA Modifications in the Central Triplet

Similar to what is described in the previous example, a number of othermodifications in AONs were investigated for efficient RNA editing usingthe GFPstop57 plasmid. For this the oligonucleotides ADAR59-2, ADAR59-10and ADAR59-22 were compared (see FIGS. 2A-2D) with controloligonucleotide ADAR65-1 and a control situation without transfection ofany oligonucleotide. In detail, 0.3×10⁶ MCF-7 cells were seeded per wellof 6-well plates in DMEM with 10% FBS. After 24 h, cells weretransfected with 50 ng GFPstop57 plasmid using Lipofectamine 2000. Afteragain 24 h, selected cell samples were transfected with 100 nM of eachAON using Lipofectamine 2000. After again 24 h, RNA was isolated fromlysed cells and used as a template for cDNA synthesis, which wasperformed using the Maxima cDNA synthesis kit using 500 ng RNA input.These samples were used for quantitative editing analysis using dropletdigital PCR (ddPCR). The ddPCR assay for absolute quantification ofnucleic acid target sequences was performed using Bio-Rad's QX-200Droplet Digital PCR system. 1 μl of cDNA obtained from the RT-PCR wasused in a total mixture of 20 μl of reaction mix, including the ddPCRSupermix for Probes no dUTP (Bio Rad), a Taqman SNP genotype assay(Thermo Fisher Scientific) with the relevant forward and reverse primerscombined with the following gene-specific probes:

Forward primer: (SEQ ID NO: 9) 5′-ACCCTTAAATTTATTTGCACTA-3′Reverse primer: (SEQ ID NO: 10) 5′-CACCATAAGAGAAAGTA-3′Wildtype probe - FAM NFQ labeled: (SEQ ID NO: 11) 5′-CTGTTCCATGGCCAAC-3′Mutant probe - VIC NFQ labeled: (SEQ ID NO: 12) 5′-CTGTTCCATAGCCAAC-3′

A total volume of 20 μl PCR mix including cDNA was filled in the middlerow of a ddPCR cartridge (Bio Rad) using a multichannel pipette. Thereplicates were divided by two cartridges. The bottom rows were filledwith 70 μl of droplet generation oil for probes (Bio Rad). After therubber gasket replacement, droplets were generated in the QX200 dropletgenerator. 40 μl of oil emulsion from the top row of the cartridge wastransferred to a 96-wells PCR plate. The PCR plate was sealed with a tinfoil for 4 sec at 170° C. using the PX1 plate sealer, followed by thefollowing PCR program: 1 cycle of enzyme activation for 10 min at 95°C., 40 cycles denaturation for 30 sec at 95° C. and annealing/extensionfor 1 min at 59.7° C., 1 cycle of enzyme deactivation for 10 min at 98°C., followed by a storage at 8° C. After PCR the plate was read andanalyzed with the QX200 droplet reader. The results of the ddPCR on cDNAobtained after RNA editing using the different antisenseoligonucleotides as discussed above on the GFPstop57 derived RNA areprovided in Table 2. This shows that in the population of PCR products,after transfection of the plasmid alone or with the controloligonucleotide, no wt copies could be detected. But, transfections withADAR59-2, ADAR59-10 and ADAR59-22 did result in significant amounts ofwild type copies in the total population. The use of ADAR59-22 (thatcarries two DNA nucleotides surrounding the C opposite the targetadenosine) resulted in RNA editing up to 8.3% wt copies. This indicatesthat use of DNA nucleotides within the Central Triplet is beneficial forefficient RNA editing using endogenous ADAR enzymes, because noadditional RNA editing enzymes were used in this experiment.

TABLE 2 Sample Mut copies/μl WT copies/μl % WT in total 50 ng GFPstp +No oligo 40,2  0 0,0 50 ng GFPstp + ADAR65-1 42,1  0 0,0 50 ng GFPstp +ADAR59-2 60,5  2,8   4,4 50 ng GFPstp + ADAR59-10 121  6,8   5,3 50 ngGFPstp + ADAR59-22 173  15,7   8,3 The number of mutant (Mut) copies perμl is given as well as the number of wild type (WT) copies per μl. Thepercentage of WT copies in the entire population is given in the rightcolumn.Hence, in a preferred embodiment, the invention also relates to AONsaccording to the invention wherein the nucleotides in the CentralTriplet are DNA nucleotides, more preferably wherein the two nucleotideson each side of the C opposite the target adenosine in the CentralTriplet are DNA nucleotides.

Example 6. RNA Editing with Endogenous ADAR on an Endogenous Target InVitro

It was then investigated whether it was possible to achieve RNA editingon an endogenous target with endogenous ADAR proteins, hence withoutover-expression of either one. The RNA encoding the mouse Small NuclearRibonucleoprotein Polypeptide A (SNRPA) was chosen as an endogenoustarget due to its medium abundant and ubiquitous expression. SNRPAassociates with stem loop II of the U1 small nuclear ribonucleoprotein,which binds the 5′ splice site of precursor mRNAs and is required forsplicing. The protein auto-regulates itself by polyadenylationinhibition of its own pre-mRNA via dimerization and has been implicatedin the coupling of splicing and polyadenylation. AONs were designed toedit the wild type stop codon (UAG) of mouse Snrpa (pre-)mRNA whichwould then likely lead to extension of the messenger and resulting in alarger protein with an increase of 25 amino acids encoded by thedownstream sequences. The original size of the SNRPA protein isapproximately 31.68 kDa and the enlarged protein is calculated to bearound 34.43 kDa. FIGS. 2A-2D show the sequence of ADAR94-1 thatcontains three DNA nucleotides in the Central Triplet, while all othernucleotides are 2′-O-methyl modified RNA. The five terminal nucleotideson either end are connected with phosphorothioate linkages. ADAR94-1that contains bulges in comparison to the target sequence outside theCentral Triplet (see PCT/EP2017/065467) was tested in HEPA1-6 and CMT-64cell lines. HEPA1-6 is derived from a BW7756 mouse hepatoma that arosein a C57/L mouse. CMT-64 was isolated from a primary alveogenic lungcarcinoma tumor mass in C57BL/lcrf mouse. Cells were plated in a 6-wellplate 24 h prior to transfection in a density of 1.75×10⁵ cells/well forHEPA1-6, and 1.5×10⁵ cells/well for CMT-64. Both cell lines werecultured in regular culture medium (DMEM+10% FBS). Cells were either nottransfected (NT control), transfected with an unrelated non-targetingoligo (NTO control; 200 nM of a 50-mer oligonucleotide) or transfectedwith final concentration of 100 nM ADAR94-1 plus a mouse specificSnrpa-related sense oligonucleotide that is thought to stabilize the AONfurther (SON2, see FIGS. 2A-2D) using Lipofectamine 2000 (Invitrogen)following the manufacturers protocol. Unpublished patent application GB1700939.0 describes the use of protecting sense oligonucleotides (SONs)to stabilize antisense oligonucleotides (AONs) in RNA editing further.ADAR94-1 and SON2 were diluted to 100 μM stocks and mixed to a 1:1 ratioand incubated with the following annealing program: 60° C. 5 min, 55° C.5 min, 50° C. 5 min, 45° C. 5 min, 40° C. 5 min, 35° C. 5 min, 30° C. 5min, 20° C. 5 min, 10° C. until use. Prior to the transfection, mediumwas replaced to 1.7 mL/well fresh medium and transfection mix (300 μl)was added, making a total of 2 mL/well. 24 h after transfection 2 mLfresh medium was added to each well. Cells were incubated for another 24h. Then, medium was removed and cells were washed once with 1×PBS andthen 350 μl Trizol was added to each well for cell lysis. The lyzedcells were then collected and RNA was extracted with the Direct-Zol RNAminiprep (Zymo) following the instructions provided by the manufacturer.It was chosen not use the on-column DNAse of this kit, but instead theTURBO DNA-Free™ Kit was used for DNAse treatment of the RNA samples. Forthis 0.5 μl of RNAse inhibitor was added to each sample, and the rest ofthe steps were performed according to the manufacturer instructions. RNAconcentrations were measured using the Nanodrop and 400 ng RNA was usedfor cDNA synthesis with the Maxima Reverse Transcriptase kit(ThermoFisher Scientific) using the protocols of the manufacturer. PCRwas performed using forward primer Fw1_mSNRPA (5′-GCCTTCGTGGAGTTTGACA-3′SEQ ID NO:13) and reverse primer Rev1_mSNRPA (5′-ACACACGGCTCTGAGAAGGT-3′SEQ ID NO:14) using methods generally known to the person skilled in theart. PCR products were checked on an Agilent 2100 Bioanalyzer andpurified with the Nucleo-Spin Gel and PCR clean-up kit (Macherey-Nagel).Purified products were sequenced with the sequencing primer Snrp-1-Fw1(5′-CGTGGAGTTTGACAATGAAGT-3′ SEQ ID NO:15).

Sequencing results for the HEPA1-6 cells are shown in FIG. 10A and thesequencing results for the CMT64 cells are shown in FIG. 10B. Clearly,the non-transfected (NT, left panels) and non-targeting oligonucleotide(NTO, middle panels) controls show no detectable RNA editing at the stopcodon (middle position of the stop codon indicated by an arrow).However, as can be clearly seen in the right panels, there issignificant RNA editing detectable when the ADAR94-1 oligonucleotide wasused (here in combination with the protecting SON2 oligonucleotide),both in HEPA1-6 cells as in CMT64 cells. FIG. 11 shows that this SNRPARNA editing can also be achieved without the protecting SON, but thatthe addition of the SON boosted the level of RNA editing. Procedures forthis +/−SON experiment were similar to that described above andperformed in HEPA1-6 cells.

These results show that RNA editing can be achieved with endogenous ADARenzymes on an endogenous target RNA sequence, in this non-limitingexample using Snrpa target RNA as a model. Importantly, it shows thatwhen the Central Triplet of the AON consists of three sequential DNAnucleotides (while the rest of the AON consists of RNA nucleotides, thatare preferably 2′-O-methyl modified, very clear and significant levelsof RNA editing can be achieved.

Therefore, in a preferred aspect, the present invention relates to anRNA-editing AON comprising a Central Triplet of 3 sequentialnucleotides, wherein the nucleotide directly opposite the targetadenosine is the middle nucleotide of the Central Triplet, wherein 1,and preferably 2, and even more preferably all 3 nucleotides in saidCentral Triplet are DNA nucleotides to render the AON more stable and/ormore effective in inducing deamination of the target adenosine. Inanother preferred aspect, the remainder of the AON consists of RNAnucleotides that preferably are 2′-O-methyl modified.

Example 7. RNA Editing with Endogenous ADAR on an Endogenous Target ExVivo

After having found evidence of in vitro ADAR-mediated RNA editing of theSnrpa target sequence as described in the previous example, it wasinvestigated whether it was possible to achieve RNA editing ex vivo withendogenous ADAR proteins in murine wild type primary lung cells usingthe same ADAR94-1 oligonucleotide (see FIGS. 2A-2D and example 6). Cellswere isolated from a mouse with a C57BL/6J background using a mouse lungdissociation kit from Miltenyi Biotec (Article nr. 130-095-927). Inshort, all reagents were prepared under sterile conditions according tothe manufacturer. Mice were sacrificed by CO₂ asphyxiation and perfusedwith PBS. Mouse lungs were then dissected from the body and transferredto a 50 ml tube containing PBS on ice. Mouse lungs were subsequentlydissected into single lobes and transferred to a gentleMACS C-tubecontaining a kit-specific enzyme mix. Tissue was processed using thegentleMACS program 37C_m_LDK_1. After termination of the program,samples were applied to a MACS SmartStrainer (70 μm) and centrifuged at300×g for 10 min at 4° C. Supernatant was discarded and cells wereresuspended in 10 ml DMEM+10% FBS, counted and cultured in anappropriate culture flask until further processing. For transfection,cells were plated at a density of 3.0×10⁵ cells/well. Theoligonucleotides were annealed to SON2, as described in example 6. Cellswere either not transfected (NT), transfected with final concentrationof 100 nM oligo targeting the human SNRPA sequence (ADAR87-1+SON2) ortransfected with final concentration of 100 nM oligo targeting the mouseSnrpa sequence (ADAR94-1+SON2) using Turbofect (ThermoFisher Scientific)following the manufacturer's protocol. See FIGS. 2A-2D for the sequenceof ADAR87-1 (see also PCT/EP2017/065467). 6 h after transfection, themedium was replaced with fresh DMEM+10% FBS and cells were cultured intotal for 48 h after transfection. After these 48 h, cells were washedonce with 1×PBS and 350 μl Trizol was added to each well for cell lysis.RNA was extracted with the Direct-Zol RNA miniprep (Zymo) according tothe manufacturer's protocol. RNA concentrations were measured using theNanodrop and 500 ng RNA was used for cDNA synthesis with the MaximaReverse Transcriptase kit (ThermoFisher Scientific) according to themanufacturer's protocol. PCR was performed using forward primerFw2_mSNRPA (5′-GCTCTCCATGCTCTTCAACC-3′ SEQ ID NO:16) and reverse primerRev2_mSNRPA (5′-TCAGGGACTGAGCCAAGG-3′ SEQ ID NO:17) using methodsgenerally known to the person skilled in the art. PCR products werechecked on an Agilent 2100 Bioanalyzer and purified with the Nucleo-SpinGel and PCR clean-up kit (Macherey-Nagel). Purified products weresequenced with sequencing primer Snrp-1-Fw1 (see above). Sequencingresults are shown in FIG. 12 . The targeted A in the TAG stop codon isdepicted by an arrow. ADAR mediated editing will be visible in DNAsequences as a G peak under the A peak in the TAG codon. Thenon-transfected (NT, left) and non-targeting control oligonucleotide(ADAR87-1+SON2, middle) show no detectable RNA editing at the TAG stopcodon. However, when cells are transfected with ADAR94-1+SON2 (rightpanel) a clear detectable editing is observed in the TAG stop codon.

It is concluded that RNA editing can be achieved with endogenous ADARusing endogenous Snrpa target RNA as a model. Moreover, as the cellsused in this example were directly isolated from mice, it indicates thatsite-specific RNA editing with endogenous ADAR on an endogenous targetusing a (therapeutic) oligonucleotide is feasible in vivo. In additionto this, and as already shown in example 6, it indicates that when theCentral Triplet of the targeting AON consists of three sequential DNAnucleotides (while the rest of the AON consists of RNA nucleotides thatare preferably 2′-O-methyl modified) very clear and significant levelsof RNA editing can be achieved with endogenous levels of ADAR.

Example 8. RNA Editing in Murine WT Primary Lung Cells on Snrpa(Pre)mRNA Measured by ddPCR

Additionally, chemical modifications of nucleosides in positions outsidethe so-called Central Triplet were investigated for further improvementof RNA editing ability. Such was also tested in the Snrpa model asdescribed in the previous examples using ADAR89-10, -15, and -20. Theribose of the nucleoside at position −1 was 2′-O-methyl modified, andthe nucleosides at positions +2 and +3 it were modified as follows:2′-O-methyl in ADAR89-10, deoxyribose (DNA) in ADAR89-15, and 2′-NH₂ inADAR89-20 (FIGS. 2A-2D). The editing ability of these AONs were testedin primary lung cells assay as described in the previous example, andanalyzed by a ddPCR technique as follows. Absolute quantification ofnucleic acid target sequences was performed using BioRad's QX-200Droplet Digital PCR system. 1 μl of cDNA obtained from the RT-PCR (1/10diluted) was used in a total mixture of 20 μl of reaction mix, includingthe ddPCR Supermix for Probes no dUTP (BioRad), a Taqman SNP genotypeassay with the relevant forward and reverse primers combined with thefollowing gene-specific probes:

Forward primer (SEQ ID NO: 18) 5′-GCAAGGCTTTAAGATCACACAAA-3′Reverse primer (SEQ ID NO: 19) 5′-GGAAGGGACTGGGGTACTC-3′wt probe (HEX IBFQ label) (SEQ ID NO: 20)5′-TTTGCCAAGAAGTAGCGCCTTTCCCT-3′ mutant probe (FAM IBFQ label)(SEQ ID NO: 21) 5′-TTTGCCAAGAAGTGGCGCCTTTCCCT-3′

A total volume of 20 μl PCR mix including cDNA was filled in the middlerow of a ddPCR cartridge (Bio Rad) using a multichannel pipette. Thereplicates were divided by two cartridges. The bottom rows were filledwith 70 μl of droplet generation oil for probes (Bio Rad). After therubber gasket replacement droplets were generated in the QX200 dropletgenerator. 40 μl of oil emulsion from the top row of the cartridge wastransferred to a 96-wells PCR plate. The PCR plate was sealed with a tinfoil for 4 sec at 170° C. using the PX1 plate sealer and directlyfollowed by the following PCR program: 1 cycle of enzyme activation for10 min at 95° C., 40 cycles denaturation for 30 sec at 95° C. andannealing/extension for 1 min at 63.8° C., 1 cycle of enzymedeactivation for 10 min at 98° C., followed by a storage at 8° C. Afterthe PCR program the plate was read out and analyzed with the QX200droplet reader with the following settings: Absolute quantification,Supermix for probes no dUTP, Ch1 FAM Wildtype and CH2 HEX mutant. Theresults as provided in FIG. 13 show that significant editing of thetarget RNA was achieved by all AONs, with ADAR89-20 performing best.

Example 9. Chemical Modifications in AONs Targeting Mouse IDUA RNA forRNA Editing

Then, the inventors asked themselves whether the modifications thatyielded stabilization could be further combined and whether the AONswith such combinations would be functional in RNA editing in the Hurlermodel (see also example 2). The effect of these AONs on restoring thewild type sequence was tested in an assay that measures the activity ofthe α-L-iduronidase enzyme encoded by the mouse ldua gene. For this,immortalized mouse embryonic fibroblast cells (70,000 per sample)derived from a W392X mutant mouse were cultured in growth medium(DMEM/10% FCS), and transfected with 1 μg of an expression plasmidcarrying the full length ldua W392X cDNA using Lipofectamine 3000. After24 h, the cells were similarly transfected with 100 nM (finalconcentration) of each of the respective AONs of the ADAR65 series (seeFIGS. 5 and 6 for stability results), and cultured for an additional 48h. Cells were then collected and lysed in mPER buffer (Thermo Scientific#78501). The cell fragments were removed from the lysates bycentrifugation and 25 μl of the supernatant was used for the enzymatic 5assay: 25 μl of 360 μM 4-Methylumbelliferyl α-L-iduronide in 0.4 Msodium formate buffer (pH 3.5) was added in the lysate samples, whichwere then incubated for 2 h at 37° C. Reaction was terminated byaddition of 200 μl of 0.17 M glycine buffer (pH 9.9), and the resultingfluorescent intensity was then measured (excitation wavelength 365 nmand emission 450 nm). Results were normalized to total proteinconcentration of the samples, as 10 measured by BCA assay (Pierce™ BCAProtein Assay Kit, Thermo Scientific).

The results provided in FIG. 14 show that all of transfected ADAR65oligonucleotides were able to increase α-L-iduronidase enzyme activityabove non-transfected (NT) levels, indicating that the target (pre-)mRNAwas edited. It appears that most of the applied AONs in fact edit thetarget RNA quite efficiently. As can be seen in a comparison with thestability assay (FIGS. 5 and 6 ) many of the AONs (especially ADAR65-23to ADAR65-30) show high stability and also show good ability to restoreenzymatic activity of the α-L-iduronidase enzyme, showing that the useof DNA nucleotides, and 2′-fluoro modifications as well asphosphorothioate linkages within the Central Triplet are useful andtherefore preferred embodiments of the present invention.

The invention claimed is:
 1. An antisense oligonucleotide (AON)comprising a Central Triplet of 3 sequential nucleotides, wherein (i)the AON is capable of forming a double stranded complex with a targetRNA molecule in a cell comprising a target adenosine; (ii) thenucleotide directly opposite the target adenosine is the middlenucleotide of the Central Triplet; (iii) 1, 2 or 3 nucleotides in theCentral Triplet comprise a sugar modification and/or a base modificationto render the AON more stable and/or more effective in inducingdeamination of the target adenosine; with the proviso that the middlenucleotide does not have a 2′-O-methyl modification; (iv) the AON doesnot comprise a 5′-terminal O6-benzylguanosine; (v) the AON does notcomprise a portion that is capable of forming an intramolecularstem-loop structure that is capable of binding a mammalian ADAR enzymepresent in the cell; and, (vi) the AON can mediate the deamination ofthe target adenosine by the ADAR enzyme.
 2. The AON of claim 1, wherein2 or 3 nucleotides in the Central Triplet do not have a 2′-O-methylmodification.
 3. The AON of claim 1, wherein 2 or 3 nucleotides in theCentral Triplet do not have a 2′-O-alkyl modification.
 4. The AON ofclaim 1, wherein 1 or 2 nucleotides in the Central Triplet other thanthe middle nucleotide are inosine.
 5. The AON of claim 4, wherein the 3′nucleotide of the Central Triplet is inosine.
 6. The AON of claim 4,wherein 1 nucleotide in the Central Triplet other than the middlenucleotide is inosine.
 7. The AON of claim 1, wherein the sugarmodification is selected from the group consisting of deoxyribose (DNA),Unlocked Nucleic Acid (UNA) and 2′-fluororibose.
 8. The AON of claim 1,wherein the AON comprises at least one internucleoside linkagemodification selected from the group consisting of phosphorothioate,3′-methylenephosphonate, 5′-methylenephosphonate, 3′-phosphoroamidateand 2′-5′-phosphodiester.
 9. The AON of claim 8, wherein the 2, 3, 4, 5,or 6 terminal nucleotides of the 5′ and 3′ terminus of the AON arelinked with phosphorothioate linkages.
 10. The AON of claim 1, whereinthe base modification is selected from the group consisting of2-aminopurine, 2,6-diaminopurine, 3-deazaadenosine, 7-deazaadenosine,7-methyladenosine, 8-azidoadenosine, 8-methyladenosine,5-hydroxymethylcytosine, 5-methylcytidine, pyrrolocytidine,7-aminomethyl-7-deazaguanosine, 7-deazaguanosine, 7-methylguanosine,8-aza-7-deazaguanosine, thienoguanosine, inosine, 4-thio-uridine,5-methoxyuridine, dihydrouridine, and pseudouridine.
 11. The AON ofclaim 1, wherein the middle nucleotide in the Central Triplet is acytidine or a uridine.
 12. The AON of claim 1, wherein one or morenucleotides in the AON outside the Central Triplet comprise amodification selected from the group consisting of DNA, a 2′-O-alkylgroup, a 2′-O-methoxyethyl group, a 2′-F group, a 2′-NH₂ group, an LNA,and any combinations thereof.
 13. The AON of claim 12, wherein the2′-O-alkyl group is a 2′-O-methyl group.
 14. The AON of claim 1, whereinthe AON is longer than 10, 11, 12, 13, 14, 15, 16 or 17 nucleotides, andwherein the AON is shorter than 100 nucleotides.
 15. The AON of claim14, wherein the AON comprises 18 to 70 nucleotides.
 16. The AON of claim15, wherein the AON comprises 18 to 60 nucleotides.
 17. The AON of claim16, wherein the AON comprises 18 to 50 nucleotides.
 18. The AON of claim14, wherein the AON is shorter than 60 nucleotides.
 19. The AON of claim18, wherein the AON comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides.
 20. The AON of claim 14, wherein theAON is 30 nucleotides long.
 21. The AON of claim 1, wherein the targetRNA molecule encodes alpha-1-antitrypsin (A1AT) or LRRK2, or wherein thetarget RNA molecule is encoded by the IDUA gene.
 22. The AON of claim21, wherein the target RNA molecule encodes A1AT to edit a 9989G>Amutation or a 1096G>A mutation, the target RNA molecule encodes LRRK2 toedit a G6055A mutation, or the target RNA molecule is encoded by theIDUA gene to edit a c.1205G>A (W402X) mutation.
 23. The AON of claim 1,wherein the cell is a human cell.
 24. The AON of claim 1, wherein the 5terminal nucleotides of the 5′ and 3′ terminus of the AON are linkedwith phosphorothioate linkages.
 25. The AON of claim 1, wherein at leastone nucleotide in the Central Triplet is a modified uridine.
 26. The AONof claim 25, wherein the modified uridine is a pseudouridine.
 27. TheAON of claim 26, wherein the pseudouridine is present at the centralposition of the Central Triplet.
 28. The AON of claim 1, wherein the AONcomprises at least one phosphoramidate internucleoside linkagemodification.
 29. A pharmaceutical composition comprising the AON ofclaim 1, and a pharmaceutically acceptable carrier.
 30. A method oftreating a genetic disorder in a human subject in need thereof, themethod comprising administering the AON of claim 1 to the subject,wherein the genetic disorder is alpha-1-antitrypsin (A1AT) deficiency,Hurler Syndrome, or Parkinson's disease.
 31. The method of claim 30,wherein the AON administration is in vivo or ex vivo.
 32. The method ofclaim 30, wherein the AON is formulated for intravenous administration.33. A method for the deamination of at least one specific targetadenosine present in a target RNA molecule in a cell, the methodcomprising the steps of: (i) providing the cell with the AON of claim 1;(ii) allowing uptake by the cell of the AON; (iii) allowing annealing ofthe AON to the target RNA molecule; and, (iv) allowing a mammalian ADARenzyme comprising a natural dsRNA binding domain as found in the wildtype enzyme to deaminate the target adenosine in the target RNA moleculeto an inosine.
 34. The method of claim 33, wherein the method furthercomprises: a) sequencing the target RNA molecule; b) assessing thepresence of a functional, elongated, full length and/or wild typeprotein when the target adenosine is located in a UGA or UAG stop codon,which is edited to a UGG codon through the deamination; c) assessing thepresence of a functional, elongated, full length and/or wild typeprotein when two target adenosines are located in a UAA stop codon,which is edited to a UGG codon through the deamination of both targetadenosines; d) assessing whether splicing of the pre-mRNA was altered bythe deamination; or e) using a functional read-out, wherein the targetRNA molecule after the deamination encodes a functional, full length,elongated and/or wild type protein.
 35. The method of claim 33, whereinthe cell is a skin cell, a lung cell, a heart cell, a kidney cell, aliver cell, a pancreas cell, a gut cell, a muscle cell, a gland cell, aneye cell, a brain cell, or a blood cell.
 36. The method of claim 33,wherein the cell is a stem cell.
 37. The method of claim 36, wherein thestem cell is an embryonic stem cell, a pluripotent stem cell, atotipotent stem cell, or an induced pluripotent stem cell.
 38. Themethod of claim 33, wherein the target RNA molecule is selected from amessenger RNA (mRNA), a pre-mRNA, a ribosomal RNA (rRNA), amitochondrial RNA (mtRNA), a transfer RNA (tRNA), noncoding RNA (ncRNA),or a microRNA (miRNA).
 39. An AON comprising a Central Triplet of 3sequential nucleotides, wherein (i) the AON is capable of forming adouble stranded complex with an A1AT target RNA molecule in a human cellcomprising a target adenosine; (ii) the nucleotide directly opposite thetarget adenosine is the middle nucleotide of the Central Triplet; (iii)all three nucleotides in the Central Triplet comprise a 2′-deoxyribosemodification; (iv) one nucleotide in the Central Triplet other than themiddle nucleotide is deoxyinosine; (v) the AON comprises at least onephosphorothioate internucleoside linkage (vi) the AON does not comprisea 5′-terminal O6-benzylguanosine; (vii) the AON does not comprise aportion that is capable of forming an intramolecular stem-loop structurethat is capable of binding a human ADAR enzyme present in the humancell; and, (viii) the AON can mediate the deamination of the targetadenosine in the A1AT target RNA molecule by the human ADAR enzyme. 40.A method of treating A1AT deficiency in a human subject in need thereofcomprising administering the AON of claim 39 or a pharmaceuticalcomposition comprising said AON to the subject.